MEMORANDUM
BRISTOL LABORATORIES

UNIT OF BRISTOL-MYERS COMPANY

 

 

 

 

 

FROM J. Lederberg DATE November 7, 1955

TO J. Lein SUBJECT_3ew Antibiotics Screening Program -
Consultantship Arrangement

CL.

Dear Joe!

We gather you have had some interesting travel lately. I've been running around a
vit myself but find the experience more exhausting than etimlating. I am going to
keep in mind the vossibility of making a visit to you before the end of the year but
T can't say just yet when it will be possible. One of the complications is that I
am teaching and have classes on Tuesdays and Thursdays. This makes it a bit awkward
for a visit which requires the best part of a day to get there and a similar time to
get back. I would be curious to know how hapry you and your colleagues would be
about a visit on a Saturday or on the Firday of the Thanksgiving weekend or similer
dates. Turing the Christmas recess or after the present semester this problem won't
be nearly so urgent.

To take up the subjects in order, of course I have seen the properdin work in the
newspapers but I hardly know anything about it but I will keep my eyes open and make
no comment until I have familiarised myself a little better with that situation.

I am happy to note that you are getting somewhere with the induction syatem but I have
the feeling that it has not yet been perfected to the point where it would be really
worthwhile to use in its present form. While, as you point out in your discussion,
you do get a 100-fold increase in the amount of phage over a period of 5 hours with
one gamma asaserine, this really should be compared to the amount of phare that you
get in similar cultures without asaserine and the cdmparisons that I see here would

be the order to 102 plaques/ml in M-9 alone as compared to perhaps twenty times that
mach in the presence of asaserine. Considering that asaserine has already been
purified and demonstrated elsewhere to be a very efficient vroducing agent this still
does not seem to me to be a very satisfactory differential,

Loeking over the protocols I would have the following suggestions to make by way of
sharpening up the system, First of all, it is quite true that azsaserine is of course
antagonized by other substances in nutrient broth. I understand that the aromatic
amino acide are especially efficient in this respect. On the other hand, I do not
think that you can get anywhere near optimal induction in synthetic media, especially
without aeration. I would have the following conerete suggestions to make by way of
modifying the technique. First of all, either to improve the conditions of growth in
the synthetic medium specifically with the help of aeration, preferably on a rotator
or shaker or something of that sort, or else to conduct the induction in a complex
medium, In my own hands M-9 is not as satisfactory a medium for the growth of B, coli
as is the Davis minimal medium which is deseribed in some of my papers and also in
Bernie Davis' but we have found that under any circumetances or any media that aeration
ia quite necessary in order to achieve maximum yields of phace. My second point would
be that it would probably be more effective in terms of induction if you could treat
the bacteria for a relatively limited period of time, say one hour or two hours, in
the presence of an inducing agent and then either dilute or, in place of that, wash
the cells so as to replace the medium with one that is optimal for growth,
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fo: J. Lein Wovember 7, 1955 Hew Antibiotics Sereening Program -
Consultantship Arrangenent

Im my own opinion the best regime would be something at follows: Use 8 hour or 10
hour inocula from a complex medium, nutrient broth, or, Penasesy medium is one which
we have used very effectively. Waeh the cells and resuepend them at «a density of
about 108 celle per ml in a synthetic medium in the presence of whatever compounds
you are trying to test the induetion powers of. Incubate with aeration during this
interval for about 2 hours. Then either dilute or wash the selle and replace them
with an aerated Fenaseny medium. Another 2 hours should be quite sufficient for the
sompletion of release of any phage that is going to be released by the induced cells.

I think with such a technique you should get, with an agent like asaeerine, levele of
induced phage at least 100 or perhaps 10,000 times higher than you are now recording.
This will of course involve using sonewhat deneer suspensions of baeteria during the
treatment. I am also concerned about the rather high basal level of the phage that you
are reporting and I suspest that the difficulty here is that you are aot using quite
enough strevtomycin in the indicator plates. I would certainly try at least 50 and
preferably 100 or even higher ceneentrations of streptomycin, that is, in gaamea per

ml, in making the assays. I suspect that at 10 gamma/ml the lysogenic bacteria continue
to preduce a small amount of phage in the assay plates but high concentrations of
streptomycin should ston them much more abruptly. With washed eells you should get a
count of pleque formers in your untreated suspension which is considerably less than one
per 1000 of the colony formers—-considerably less. 1 would imagine that the results you
are getting now would be pretty much the same as if you were not adding atreptomycin to
the indicator medinun,

I don't believe thet we will have any further or additional use of a W1h-89Sr strain at
the present time but thank you anyhow, Joe. I do think, however, that you might find
it advantageous to leok into the use of a few other strains of sensitive basteria, for
example, strain C, which I believe we have sent you before.

The considerable variability in plaque sise that you mentioned is not uncharacteristic

of random platings. It's one of the serious headaches of this partieular system and

I don't know too well how you are going to get around jt. I suspect that the plaque
variation comes about partly from the fact that through a plating, both free phage particles
and infeeted centers which are shortly about them in these very large numbers of phage
particles, the variation may disappear or at least be greatly reduced with the regime

that I have mentioned before or with the incorporation of more atreptomyecin inte the assay
plates. Something else might conceivably be happening, viz., that your stock culture has
picked up a certain amount of a mtant to phace which we call lambda 2 which is capable of
growing lithely on the K-12 strain. You can rather easily check to see if any of the
rather clear plaques that you have in your assay plates are thie mutant by simply
determining whether they will lyse the K-12 strain itself. If this ie the trouble, or in
fact even if it ien't, there might be some advantage in asing as a test culture ea mtant
of K~12 which is resistant to lambda 2 which mtation did not interfere at all with its
ability to release lamdde under conditions of induction and we will send this on to you in
awhile. This is the so-called lambda 2F or LPo¥ mutation. If I may summarise the
following are pointe which I think wll contribute to giving you a much sharper assay than
you have now:

1. Conduct the treatment of the bacteria in a synthetio medium but subsequent to a very
brief treatment they should be allowed to develop and grow the phage in a complex
medium under conditions of aeration.

2. Use considerably higher levels of streptomycin in the aseay plates.
3

To: J. Lein November 7, 1955 New Antibiotics Sereening Program ~
Consultantship Arrangenent

3- See whether the extreme variation in plaque morphology is due to the presence of a
lytic phage which will attack the X-12 strain, If thie is the Gase, it may partly
account for the high levels of contrel phage that you are running inte.

In my own opinion it would not be profitable te proceed with the testing of new agents
until you have a system which is rather more sensitive to an agent like azaserine,

To turn to some other iseues, I am very pleased to learn that you will have some
continuing interest at all in the problem that is related to the use of the Serratia
strain that we sent you. When you said you would feed it to see if it becomes
established, I hope you mean to give the red bacterium some encouragement. I would very
wuch doubt that it would displace a pre-existant entoric flora in the mouse. I would
have some hope that it might de so under conditions where for example you used a
streptomycin-resistant autant for the feeding at the time when the animals were
receiving streptomycin and their normal flera was being rapidly diminished. Almost
anything that comes out of such experiment is bound to be, te my mind, extremely
interesting.

TI would fully eeho the concern that you feel that the most interesting and novel
situations in the new antibiotic program might be lost in the shuffle. I think the whole
implication of what is meant by the cream having been already skimmed off this situation
by the large companies means that you have to scrounge around with somewhat more
difficulty in the skim milk. There is always bound to be at least some inverse
correlation between uniqueness and quantitative facility. I know that you are not going
to worry much about your situation until you learn whether or not Felix will be staying
on with you. I think you will be interested to learn that Tom Nelson hae taken a job
in the Botany Department here working with Fulton Skoog on the so-called lakes project.
As you know, the development of blue-green algae blooms on our local lakes has been a
matter of very great economic concern so that there has been a very extensive program
of the study of the physiology of theee organiems. Tom is going to be involved in the
development of teehniques for the mass culture of the blue-green algae but one of the
mala preocenupations or end practical results of this Bake project is sertainly going to
be the development of means of inhibiting the growth of these organiems. It just
oceurred to me that if quite the right antibiotie agents could be produced which would
be effective enough to make it worthwhile to use them on the tremendous scale recaired
here, there would be # very large market for that kind of antibiotic, that is to say,
algecidal agent. Of sourse, ené shudders to think of the ecological upsets that would
be the consequence of auch interference in the normal cyole of lake growth. At any rate
Pom ie quite well fixed up here, at least for the academic year. There is no vromise on
either side as to whether he will continue and in the event that Felix doee leave I
think you should certainly leok to see whether he is available and suitable for a
position in your outfit. On a long term basis however, as long as the subject has been
brought up, I would think that Galen Bradley would be a more effective member of your
researeh group. However, I am not going to recommend him to you or vies verses unless
your company policy would be such as to make it possible to assure Bradley that he could
spend at least the larger part of his time on basic research and I realize that this may
be much-too-much to ask at the present time. Still I don't know how you are going to be
able to get the best possible personnel to handle even the genetic application of
actinonycete genetics unlees you can make your openings attractive on this basis a8 well.

There is not a great deal to tell you about the progress ef our work here, clearly
Simonti has gotten a good deal ahead of anybody else. I think his mere demonstration
4

fo: J. Lein November 7, 1955 New Antibiotics Sereening Progran
Consultantship Arrangement

of the existence of recombination in actinomycetes ought to be sending thoughts into

the minds of a great many people. Galen had gotten about as far along technically I
think as Simonti did. We have developed a fairly good colleation of auxzotrochie

matents in various strains of streptemycetes which were ieolated from the soil with
reference only to their convenience in laboratory use. The technique that was used

for developing these auxotrophes was imply that of replica Plating of the sporulating
colonies after UV irradiation of spore suspensions. As far as we ean tell in the species
that we have considered, the conidia are strictly unique nucleate, Combinations of
different auzotrophs under conditions of rather stringent selection do result in
ocoasional prototrophs being formed, This is about as far as it had gone during ay
earlier essays into this partioular area. Galen has gone over most of that rather more
carefully and reeently we have been able to prove unambiguously that these prototrophs
are in fact heterokaryon because when single hyphae are isclated with the micromanipulator
they give rise to cultures which are prototrophic themselves but which throw off
exclusively parental auxctrephic spores. In the vegetative state, however, heterokaryotie
mycelium, onee it is formed, is relatively stable as such and this does leave the door
open for the possibility at least of exploring useful applications of heterokeryons.

So far we have not run into anything that we could be sure was recombination tn a way
that Simonti dees in the path he has done so easily so this must be very largely a
matter of atrain difference and it would be impossible to predict whether such
recombination phenomena would occur in which strains.

The only reservation that I continue to have in discussing these actinomycete problems
ia that you not tell me anything that you would not want me to pass on to Galen because
I obviously cannot hold him to any confidence on these matters if other people ask

about them but I think we've gone over that question sufficiently so that we know pretty
well where we are. Of course I would be delighted to hear whatever Felix has to say
about his own genetic program. I can certainly sympathize with the almost necessary
slowness with which it proceeds. We've had our own experience with that. Hew, by the
way, is Ethelyn Lively working out? I hove she is an asset to your organi sation,

Well I suppose the most important immediate question is the teehnique of induction and
IT hope you'll keep me informed as to whether you consider it werthwhile to try some of
these modifications and how they de work out. I'm really convinced that you can do a

lot better with the system,

By the way, my suggestion on the use of bleod azar was not quite along the lines that
you seemed to have implied it for. There would of course be no partioular advantage in
using blood agar as compared to autrient agar for simple quantitative assay of plaque
titers, It turns out however that the mere process of lysis, that is to say, of
induction, appears truly as the hemolysis and I have thought that you might be able to
get a useable measure of induction simply by adding some blood to the tubes in which the
induetion was taking place and seeing the extent of hemolysis that went on there. This
would make it quite unneesssary you see to do further platings in order to count the
number of phage particles that would be released, Whether this is stil] worth a trial

or not, I don't know, but I think it would be amusing if you would simply de an
experiment along the following lines. Grow some K-12 in complex medium, wash it, treat
it with UV light, and I would say a dosage equivalent to let's say 60 seconds of a 15
watt sterile lamp at a distanes of 50 em and then put the washed cells back inte Penassay
medium under conditions of asration together with a certain amount of dleod. You, of
course, will have a control using cells which have not been exposed to BY light and then
after a couple of hours sediment out both the bacteria and the red cells and see how much
hemoglobin has been released as the spernate. I've never done this exveriment in Just
fo: J. Lein November 7, 1955 New antibiotics Screening Program -
Consultantship Arrangement

this form but my prediction is that, correlated with the induetion of the bacteria,

there will be a substantial release of hemoglobin which could be looked for colorimetrically.
Of course it would be impossible to do a test of this sort with broths that were themselves
hemolytic, but it would seem to me that an indireet test of thie kind might let you do

awey with the rather complicated plating procedures which are needed for counting the

amount of phage which is released by induction. I am by no means certain what this
hemolytic effect actually is. I suspect it refleots the fact that the bacterial lysates

are themselves at least for awhile hemolytic but this is a question we really should

examine somewhat more directly. And we may conceivably do an experiment along these

lines ourselves at some future date.

Geedbye for now.

Joshua
SLi jle