MEMORANDUM ‘NOTED

MAY 5 1955
BRISTOL LABORATORIES A. Gourevircss

UNIT OF BRISTOL-MYERS COMPANY

J. Lederberg April 10, 1955

 

 

FROM DATE .
J. Lein Wew Antibiotics S P -
To SUBJECT_oouguttantehte Areceee ee eree
Cx.
Hello Joe:

This is dated April 10th and ia im reply to yours of April §. Your record was
largely in the nature of commente on my own and leaves me not a great deal that needs
revly. I am «lad to have the information and some of the references that you did
give me, and thank you for it. As this consultation goes on I get the feeling, and

I should be surprised if it weren't so, that I am getting a closer and closer under-
atanding of whet you're about. Of course the time will come when distance will

reach the point of diminishing returns and the very fact of being close to your
problems may somewhat diminish my usefulness but of course I'1] do my best. I hove
you won't take my every suggestion too seriously. Your deseription of my function
aes gadfly ia, I think, more appropriate than that of a delphic auricle. I wae a
little surprised therefore that you paid very much attention to the notion I
indicated of leeking for high production of accumulated intermediates ae a first ater.
T mentioned this only as, so te speak, a logical sequence, well understanding the
probable technical barriers. However, I'm going to continue to mention things which
te ay mind appear to be technically impossible because it seems to me that my function
is not primarily that of technical evaluation; that is really yours, If I took it

on myself to decide beforehand thet something wae or was not likely to work I might
be depriving you of ideas directly or indirectly which might lead to some useful
application. In this particular case the fact that the lew production of antibiotics
seems to crecluce that looking for them in the first instance by any method other then
their biological activity - well that seems very reagonable but I would still keep in
the back of my mind the notion of how worthwhile it would be to have. some independent
method of identifying such subatances.

I had a chance over the week-end to leok again at vakeman's more recent opus of
Actinoaycetes and Their Antibiotics and was of course greatly impressed by the large
mumber of antibiotics that have already been described. It strikes me that your main
problem must be not so much the discovery of anything new but the verification that

it is, in fact, new. I was not very deeply aware of what you had in mind by way of
your punch card system for the classification of your antibiotic broths but it would
seem to me that you do indeed face a difficult vrodlem in simply assessing the novely
of anything new that does come up. This is one of the things that I will want to talk
to you about in greater detail when we oan get together personally ae it sceme to ne

a rather too complicated affair to go over at thie dietance.

Aa to the general question of the natural function of antibiotics, in a way I night

say that I am in agreement with your own position on the matter that it really is

an open question. I have not had enough time in the game to have any feelings in the
matter, ag you recognise yourself the arguments that you presented are by no means
conclusive although they do point in the direction that you had incicated. I have

been particularly struck by the statement I've aeen in several places that antibiotics
tend to be adsorbed onto the soll. If the usual methocs of testing antibletic activity
would require extraction of the antibiotics tnis might fail whereas the adsorbed
2

o: J. Lein “2s New Antibiotics Screening ‘rogram -
Consultantaniv drrangemen’

antibiotics etill conceivably might function in situ under certein conditions. As to
the pointe of the low initiel activity and the epecific requirements under laboratory
conditions one could revly rather facilely, I don't know how correctly, that these
notions apply primarily to laboratory circumetencees and what goes on in the soil might
be a wery different matter. It might alac be to the point that the actual) amounts of
natiblotic required for effective cometition as opccsed to complete antageniam may be
relatively small since antibiosis is by ne means the eaclusive mechanian of auch
competition. Uncer certain circumstances one may even imagine that over production of
en antibiotic could be deleterious uncer natural conditions to the organiem which is
producing it. In fact, one can argue that this is probably true from the very fact that
it recuires laboratory selective procedures to develop high oroduction hy Actinomycetes.
Thie eames point of possible self inhibition on the one hand and the sudtility of
ecological conditions in the sei] on the other, I think, ean also be used to counter
your final point thet the antibiotics of limited enectra would be lees likely to be
ueeful to the Actinomycetes. Ee all that as it my, this is obviously a rather subdtie
euestion. It may not have wuniform anewer, that ia to say, some entiblotice may be of
some use to the organism under natural conditions and other not. As to whether they
are the blocked intermediates of normal metabolites however I am rather sore skeptical.
I have been for a long time a very firm follower of the general notions of comparative
biochemistry to the effect that one is very unlikely to find important metabolites
which will] be unique for any aingle class of organieme, If, for example, strertomycin
were an intermediate in the synthesia of a generally important metabolite I would imeine
that it would have been picked uc before now in some completely unicue clisas of
orgenieme and ae far ae I know, for examole, I think venicillin hes not been picked up
ag a product of sctinomycete mataboliam and cenvereely for streptomycin and the molds.

And flnally to finish up thie tepic for the moment agsin, have you seen the Sympestum on
Actinomycetes which waa held at the Internstional Congress of Hicroblolosy at Rome is
1953? There are a number of capera in there which will be of mutual interest and I
would emphasize at the oresent one by Villemin, Lechevalier end vakaman which goes inte
somewhat more detail on the produetion of antibiotics in the esil and where they suote
some of their own exverimente, for example, that en ether extract of soil had croperties
similar to those of Actinomycin. dowever, ae I think should be emrnasized, there fe
nothing contradictory between the blocked intermediate hypothesi«a and the ecological
function as sntibiotics, aince these concents may be looking at the eame ~sterial Just
for different noints of view.

The main thing that dees concern se on the viewpoints thet sntiblotics are blocked
intermeciates of generally important metabolites fe then why are Actinomycetes anomrently
so uniquely important ae potentiel sources of new antibiotics? They were seized upon in
the first place, I imagine, because of their crevalence in soil and more likely perhaps
because they happen to work. I wonder, however, if your main interest is the question
of finding anterials that are new and untried why you don't leawe the antiblotics
altogether and go to screening other kinds of soil organiens. This would be the noet
likely method of nicking up materials that have hitherto been ignored on the one hend
ang on the other your hypothesis of the nature of antidiotice leaves nothing unicuely
favorable for the Actinomycetes ae potential sources of them, From the proint of view
of laboratory senizulation, organiems ench as yeast or bacteria would be far nore
convenient; there would also be a rather more cogent possibility of deteiled genetic
anolyeis and cerntrol of the results. ‘The cuestion aay be how desply you are convinced
of thie hypothesis of the natura of the antibiotics anc of course in an industrial
everation !t is not necessary to commit all of one's investment to a single theoretical
Tine of aporosch. I ao feel the more strongly the longer I think of it however thot the
To: J. Lein -~3- Hew Antiblotics Screening Program -
Consultentehip Arrangsnent

most likely technique for obtaining hitherto ignored anterials would be to develop
Organians completely out of the range of the Actinomycetes altogether. If they have
veen so thoroughly screened on a random basis it would suggest that it may perhaps
be time to leok for antibletic activity from quite unique sources. Otherwiee I
woald appreciate your views on juct why Actinomycetes are euch organises of choice.
Bow 1 do appreciate, thie ie a point that has startled se in my own experience, that
the unique thing about Actinomycetes may not be so much that they are capadle of
synthesising antibiotics but that they have the habit of vroducing large amounts of
materials that they put into the medium. On the other hand I am net certain that thie
is something that properly could mot be shared by quite a wamber of other organisms.
It might be of sore interest to test Actinomycetes more directly for a trait of this
kind in an objective way, for example, by obtaining on Actinomycete which is blocked
ia some particular step syntheeis of some amino acid fer exemple and seeing whether
ite level of production of the intermediate without any further handling of the
orgeniem is going to be strikingly different from a comparable mtant of Neurospora
or bacteria or yenst.

Another logical conclusion on the notion of the ecological insignificance of antibiotic
production is that one could infer from this that there ought to be no particuler
correlation between this trait and other characters whoee persistence in natural
populations presumably would de a matter of soce ecological importence, that is to
say, I sense without verhaps being able to state it in a completely logical, rigorous
fashion certain contradictions between this notien and the idea of the necessity of
Yeoking through unicue kinds of Actinonycetes in hovees that this is the beet method

of finding new kinds of antibiotics. The blocked intermediate notion would seem to

me to lead to the conclusion that any isolate which is not of immediate direct fidshis
with any other hae an equal likelihood of produging eome new compound and I fing in
thig paver by “aleman some justification from thia point of view in the great verlety
of compounde which have been croduced by what has been classified ae a single tyre of

Streptomyces sriseug,

At one point in your commentary you ssked me to explain more carefully why I thought
too rigorous selection was bad. I think a little bit leter on you expressed the
polat very well yourself, namely, that if in your elective isolation crogram you bored
tee etrengly on 2 single carbon or nitrogen source you would, of course, before very
long be exhausting the total number of types which could be isolated on that source
and then be reduplicating your efforts. That's all I hed in mind. The more rigorous
your selection the sooner you are likely to exhauat the existing rance of organiers
that could be picked up on a given medium.

In some eerlier paragraphe I did mention the possibility of autosensitivity ae 2 factor
whieh may limit the natural production of antibiotics. I think this is something

that may have to be looked out for in ecreening for mutante which will produce
aotiblotice. I was struck, Sr example, by Kelner's comment that many of the antibiotic-
producing mutants thet he observed did in fact forz rather limited colomise and crew
rather poorly generally and it seeas to me possible this miy be due ta direct tnbibit ion
by the antiblotic produced although of course it is equally poseible thet this ie the
primary effect, namely, the metabolic defect of which sntibiatic preduction Le a
secondary consequence. This any suggeat however other methods of going efter mutants
with entiblotic-producing traite, that in to aay, to look for self-inhibitery mutante.
Tot J. Lein oboe few Antibloties Screening Frogram -
Consultantship Arrangement

Finally, to answer a couple of Dr. Gourevitch's questions, I heoe in the first place
that he hasn't expected more of replica plating for Actinonycetes than it can furnish
for any other organism. Of course, when the velvet is pressed down on a colony of
any kind of orgeniem it picks up not just a single point impression tt e emall area
and devenading on circumstances and the organiem end conditions etc. there is deund to
ve a certain amount of spreading of the growth from the initial size. That means that
even with bacteris it is necessary to limit the total population on the plate to
something of the order of 100 or 200 colonies if the colonies are sodernately emall

and to ride with less than that if they do not develop to a very large size. With

the Actinomycetes, however, we have had no trouble for a long period of time vith
replica plating snd sporulating colonies for the purpose of obtaining nuzxotrochic
matante and I can hardly think of any aituation whose demands would be sere rigorous
than thet. The main thing I cam suggent in to make your replicas as soon as 211 your
colonies on the plate heve begun to sporulate and in that way they co not get too
large, aot to press too hard with the velvet since only » very light contact ie ali
thet is neceasary and sake sure the plates are moderately dry when the tranefers are
wade. I will aleo take the occasion to discuss « little more closely the role of FAY
a8 a metabolite and as an anti-metabolite. The former is, of course, very well
recognized and the latter has been rather cbscures it is not et thie moment clearly
known what the anti-rickettsial funetion of FAV ia based upon. I can't help but feel
however that this is an utter coincidence that the two functions have nothing to de
with one another. Perhapa the strongest possibility that I am aware of for the anti-
rickettsial function of PAY is that it functions as an ataconiat of another vitearsin
that Bernie Invie has picked up, namely, hydroxybenzoic acid. At least I know that he
hoe made that suggestion and I'm not well enouzh aware of the current litereture to know
vhether it has found a better or worse basie since he first made it.

Well, Joe, I'1] be quite ready to get down to brass tacks or to make any kind of
discussion that you would like to do. I realise that things aay get to be a little
cisorganized and will vcrobably get more eo if we go on to personal discuesion. I hove
not to be belaboring any particular point too often aince wy function here is to serve
you rather than to convince you of the correctness of any conclusion tant I any have come
to and I want to be sure that I do the foremost moet effectively. “hat I will do, I
think, is collect the various and odd notions that perhens have not been dissolved

over a period of several months at a time and rerhave every 4, 5 or 6 months I'11
prepare a fresh resuse in order that you will hove these iesuee readily st your discosel
to act upon ae you see fit. I aporeciate that there are necessarily differences in
lines of action in your set-up as compared to what we would do in a laboratory like our
own anc I'm not well enough acquainted with how you can run things to be ab’e te inter-
pose my own Judgement on what you ought to do. As you suggest, however, ey function
wil] be to remind you of the things that ought to de thought about. I hope you never
thought thst a consultantship would sean that you would have someone elee do your
thinking for you. I think you are going to find the contrary; you're just going to

be faced with so many more problems tenn you had before because your coneual tants

never go into making decisions for you end his advice ia bound to be of a rather

oblique nature and as you suggest verhacs his main function is to help keep you on the
ball which means that much wore vork on your cert. Vell I hope I won't have to make

too many more ohilosophical justifications as we go on. SNowever, barring theee

rather infrequent resumeg which I will make as my undertancing of the situntion develone,
T'm going to leave it un to you to take the initintive as to which sarticuler tonics

we ought to explore in many details as time goes on. Chviously we could very readily
#0 #11] over the sap and we have to have some sort of rhyme and direction to the story
which I'm going to leave for you to do until you cive me the cues as to just what things
fo: J. Lein -5- Hew Antibiotics Screening Program -
Comasultanteship Arrangenent

you want me to go after.

So long for now. If I sound just a little bit tired I hope you'll keep in aind that
thie is the beginning of the spring gardening season.

Josh
JLsajl