November 2, 1952
Dear Bruce:

Although your reply to my last letter may well be in the mails, afew

things have come up that seem to warrant your inmediate attention.
was

The behavior of the 1,2 phase from SW-534--SW-588 warm very perplexing
to mo. If 534 came directly from 53%, as eteted in the stock list, and 533 =
Edwande' diphheic 8. paratyohi B #3, selection by Chi phage had a remarkable
set of effects: a} pleked out phase 2 (not incredible by chance); b) fixed the
strain in this phase and e) so altered 4+ as to allow its trammission to and
manifestation in SW-543 transinductions. In my recent experience with ie)
(= SW-703 by present numbering), its second phase readily varies back to b,
and ie not traneducible to SW-543. This made me suspicious of the purported
pedigrees. You will recall that we had not syetenatically numbered immigrant
serotypes until about the time of yow arrival (I should long Age have set them
up in our unfform list, as we discussed). My notes on the origin of sW-534
mention only “paratyphi B, non-motile, selected by Chi"-- I might very well
have taken the culture now designated as SW-546 (or better, SW-857 = Edwards 157).
As SW-546 does appear te be monophasic 1,2 it is a more logical candidate for
the patemity of SW-554, if this can be cast in doubt by any means. (In addition,
FA (S¥-546) -x SW-543 geve 1,2.) Some further tests seem to bear this out.
SW-703 differed from SW-546 and SW-588, and the datter aro similar in the follew-
ing: fermentation of rhanmoee and iansitol, intensity on EMB mannitol, and suegep
tibility to Chi phage (Boulgakev original), and to the phage earried by Sw-543.
I am checking with Norton to see if his notes cerry any additional gleanings,
and with Edwards on the histpry of SW-857, but thie revision is fairly certain.
If se, the 1,2 phase carried by it 46 quite unique in ite transducibility to
SW-549¥ and the notion that monophasicity has to do with an inability to mani-
fest the second phase phenotype in certain genetypic backgrounds is still tenable,
with this as an feolated exception. .

A eecond point that surprised me considerably ie the presence of a b (7)
antigen in SW-543 ( #603 = 666) line. Ite manifestation is rather irregular,
but indubitable. Inmotility 1s not impaired-- the most reactive suspension was
an inoculum in Penassay from a stationary blob in motility agar, and which
did not, budge when rainoculated to motility agar. I could not convince myself
of a trace of active motility by microscopic observation, but there is always
seme uncertainty about this. Have you ever done a flagellar stain on this one?
I'll send a culture to Leifson, and have some electron micrographs taken here.
In view of eur genetic results on the separability of antigen and flagellum
loci, we might perhaps expect the production of H antigen independently of
the latter. Don't Pijper and some others believe it to persist on the cell sur-
face, anyhow? Until an antigenic analysis is done, I can't tell whether the
complete b antigen is present-- or whether the whole thing is aome sort of uxmt
artefact.

The pedigree of progeny tests I outlined previously is almost completed: I
will review the relevant section on the reverse sheet. I have still to complete
the test of « back-X to SW-666 of an f-1 4; the difficulty was the resistance
te PLT-22 of most of these, but I have one going now that I knew to be sensitive.
In fact, it 1s rather remarkable that these serial transductions should be pos-
sible, and the pedigree already shows some lines with several steps in which
the transinductions remained susceptible to the transducing phage. This means
that the panétration of the phage (with delivery of its contents) may be

followed either by lysis, or by lysogenicity (7), or by the recurrence of
sensitive. Many of the tranesinduced swarms are, of course, selféplaqued.

One speculation hs that the phage itself may attach to & different nucleus

than its FA does. I will have to check further on the establishment of lyso-
genicity for PLT-22 in SW0666. I have the impression that the i transinduc-

tions may be more regularly resistant (lysogenic?) to PLT22(4n all the above dis-
cussion, adapted to SW-566) while the b's are sensitive, but will have to

check further.

A few more experiments on the efficiency of transduction of Galt and H+ to
SW-666. Unfortunately, there 1s by no means a linear response of Gal+ to increasin
FA, while, as you know, H+ seems to go up pretty well within the testable range.
Qne supbanty, that out to be checked: with the dame combination (FA-703 -x 666) and

dilution, at 30° there were 3 swarms, no tracks; at 37° 3 tracks, no swarms, At
any rete, the effect of environmental factors such as temperature on the TysS
choice will eventually have to be studied (2 regard this as your territory, and
this as enly a casual excursion, may I add). In some of these experiments, there
were some well isolated swarms, with very well developed flares (the track cluster:
of some hundreds of micrecolobies}, and I thought surely to be able to recover
the postulated 0. About 60 tests all told, all H (and antigenically uniform; as
expected, each swarm is pure) | Together with your results, I just don't think
the flare microcoloniea are 0, and another splanetion will be needed. Perhsps it
is just that the newly formed H's are relatively weak and unexercised; a close
look at the tracks, and the time relations conputed from oxpected division times
shows they are not moving at the final high rate, either. The full development of
motility might be either a phenomic precees, or accumulation of polygenic modifier
but I don't see that the flares can represent a segregation of clear cut O's.
This still leaves the tracks, but if they don't have to be correlated with flares
they may be celle which, as previously postulatod, adsorb E+ pheze, but in which
the entering H+ factor never does get iuto the chromeseme, whether or mot the
cell {a lysed. For the analysis of the flares, it would obviously be desirable
to have a clear cut selective technique. I've been playing with some angles oh
this-- especially Archer's method or sone variants. It was in the cource of
reconstruction experiments with SW-6035--666 and SW-618 that the b-agglutinability
of the former showed up, and of course the rather negative results I had been
getting are indecisive. SW-545 is, unfortunately, essentially resistant to Chi
Phage, but I did some reconstructions with SW-568 (Gal~wH+)and sw-666 (Gal-H-).
Starting with about 100:1 +:-, she survivors cf Chi on agar are about 1:1. (This
does not necessarily mean i Qautant/100 originally +, as the action of Chi is

by no means immediate.) Unless the selection 4s much sharper in liquid mediun,
which it may be, the method will be probably too messy to be of much help with
the flares. (But I don't see how the microcOlonics could have been passed over

in 60 tests anyhow!) Attempts to dilute out the H+ proferentdally bytheir spreadin
into non-nufrient soft agar were wueuccessful.

Perhaps the moet interesting developments are some rather sketchy fates on
which to hang a theory of phase variation. Abony and tychimurium (L™2) have made
a ataisfactory combination, although it would have been amusing to have had differ
ent somatic groups as well, In both our experiments, barring SW-546..., the PA
from phase 2 has shown no trace ef the phase 1 component, ¢+g. in transduction
to SW-543 or to typhi. This holds us well for enx and for 1,2 of the present
material. However, FAC bienx)--x 4,12 gave b:1,2 (selection by 1,12 serum), and
btenx —X 1,12 gave itenx. That is, only one phase ia traneduced, the other is
latent or residual in the transducee. This excludes the idea that the phases are
simply alternative alleles, and suggests that there are two loci, one for spe-
cific alleles, the other for non-specific. This would fit very well also with
the patterns of phase variation in the group. ‘he paradox is that the alterna-
tive phase seems to be latent in the cells as transducees, but not as trans-
inducers. Qne can now either make very special asshmptions about transduction
in general, or about the genic mechanism of phase vurlation. On the latter,
we infer that a “cytoplasmic state" detewmination of phase is excluded. Nor
can we accept my old "awitch factor" hypothesis, as the factor should be
separable in traneduction from the loci it controls. I am left with a general
notion ef differential,(and mutually exclusive ae between the +3 loci) gene
states: i.e., the activator (or inactivator) of the loous which is expressed
phenotypically in the zhass antigenic phase is inseparable from it. On a par-
ticulate basie, this is analogous to McClintockés fia factor in corn, but we
could just as well think of itg in physiological, albeit self-perpetuating,
states for which there are innumerable possibilities--E.G. Huskins lateral
reduplication. If transduction can use the cytoplasm, we could even drag in
reduplicated plaemagenea ef the kind that are fairly closely dependent on the
locue. The tranaductions from O-forms fertumately relieve the antigens them-
selves of this genetic burdeyn-- but all the more reason for making sure ximt«
about the wnournnw complete absence of H antigen from then.

 

Before long, I will nave to submit abstracts to the International Congresses.
I heave a formal invitation from the Gentics Congress (Lake Come) and have
reason to expect pas another fren the Microblologiste (I was already asked to
speak, of all things, on actinomycetes!). I assume you are going te beth Con-
gresses yourself. I think it would be less complicated to avoid joint author-
ships, although we should censult with each ether to economize on time. The
Genetics paper will be a 2C~30 minute affair, and probably a rather general
review of geastic wocheniens in bacteria in general (that is te say Cold and
Salmoneilat) This showld not conflict with’? Sresentation of the work with
motility andneductions which might as well he under your sole authorship. I
don't know yet whether Norton is traveliing alse, rather expect not unlese

he gets a windfall (we can say the apte, for that matter!)

With the coupletion of the pregeny teats’ vary close te hand, I don't see
what remains now to be dons t should postpone writing fp this work. 2xingws
khatxgom What explicit po are now ovidant?y that need to be included? I
hope you can find on time to do this during the next few weeks-- if it
would help I would be #illing (but not eager) te go ahead on the basis of the
outline @Hat you wrdte up before leaving here. In any event, the authorship
Stocker-Lederbeyg-Zinder should need no further discussion, nor, as I would
imagine to be-your preference, its preparation for the Journal of General Micre-

biology. you put together all the rest of 1t,—T will -sellect my data on _
———thie § eny tests and add them to it; a chart would probably be indispensable.

    
 

 

Sincerely

Joshua Lederberg