August 2h, 195) Dear Professor Lederberg : Thank you for your letter of August ). I am very glad to hear that you have been appointed as professor of Genetics. Allow me to congratulate you 1 I wish to express my appreciation for your kind suggestions and inquiries. 1 will do my best to answer, And I do not know how to thank you enough for correct- ing my papers and even offering to read the galley proof. I could ask for nothing better. Unfortunately I had an acute attack of appendicitis and have been hospitalized. I hope you will understand the delay. Thanking you again for everything. Yours sincerely, Hitae (take Hisao Uetake Dept. of Microbiology Sappro Medical College page 3 "\". By broth I mean the usual nutrient broth (bouillon), Mixed cultures were incubated at 37°C for 2l; hours. They are standing cultures, not aerated. page 5 "9", The phage suspension used for enzymatic treatments was not an autolysate preparation itself. It was a high titerjstock, prepared by propagation by serial transfers of phages on S. anatum. page 6 first paragraph: When found, alteredcolonies were found at a rate of one or more out of 20. “It was confirm- ed that all of the autolysates were capable of form- ing plaques on S. anatum or S. butantan, though the exact titrations of all the autolysates were not carried out. Serological comparison of the phages and comparison of their host ranges are now under study. page 11 VIII. Even after one minute exposure, one or more out of 20 colonies were found to be agglutinable by 15 antiserum, TABLE 3Rate of phage-sensitive and mtigenically altered cells, One minute exposure is too short to carry out a quantitative determinationg of phage-sensitive and antigenically altered cells. Then, the quantitative determination was carried out after 430 minutes ex- posure and the following is an example of data on this series of experiments. Dilu- Noe of io. of No. of altered tion | total translucent| colonies among colonies colonies O non-trans~- ucent colonies Active phage P(5%108 particles/ml) 1;104 | 16 20 33 + e S$. anatum (108 celis/ml) 1:105 1,0 2 21 Heat~ina ctivatednphage ( same as’ above) 1:106 10h, 0 0 + * S. anatum (same as above) 1:107 7 0 0 The phage particles (from S. canoga) and the Ej group cells (20-hour-old agar culture of S. anatum) were mixed at the rate of 5:1, kept standing at room temperature for 30 minutes and pla ed out for Vv able count. : uvVl ivi Q colonies were also tested serologically. io a x Mean number from 5 plates This experiment revealed the following. (i) About O out of 700-100 cells survived after exposure to phage. (411i) About lO to 60 per cent andng non-trans lucent colonies were of antigenic variants. (iii) All of translucent colonies were found to be agglutinable by 15 antiserun, 2) and 3) that you have been kind enough to point out in the letter have not been made clear as yet. (Thank you for your information on translucent colonies. I am not as yet acquainted with your paper in Genetics, since it is not available here. Theugh translucent colonies have been found to contain anti- genid/variant,non-variant cells, other-eetis, other details are nSt clear as yet. The details will be studied further.) page 9 In each of antigenic variants 2 or .3 . isolations were tested for lysogenicity and conversion ability. page l2 As biochemical behaviors the followings were examined, In peptone broth medium, fermentation of glucose, mannitol, dulcitol, sorbitol, inositol, maltose, arabinose, rhamnose, xylose, trehalose, adonitol, salicin, saccharose and lactose, In Bitter's medium, fermentation of arabinose, dulcitol, glucose and rhamnose,. Beside the above indole production, gelatin liq efaction, milk coagulation, decomposition of urea and acetyl- methyl-carbinol formation were tested, page 13 I agree with your opinion "en should be corrected to diwection of antigenic changes found after exposure to antiserum. page 1, "l)". The experiments for the statements ")h)" were as follows. rt A small number of phages from gs, canoga were added i” to nutrient broth in test tube. In this phage-breth mixture S. anatum was cultivated at 37°C for 20 hours, The bacterial cells were centrifuged, the supernate was removed, filtered through Chamberland Lz filter C and the filtrate was added to broth culture’ of S,. anatum which had been incubated for about 8 to 10 hours. After further incubation for 3 to l hours, phage suspen- sion was obtained by centrifugation and filtration and phages were propagated again in the same way as described above. These processes were repeated through several serial passages, and finally phage suspension of relatively high titer was obtained, containing about | 4 x 107 particles per ml estimated by plaque count. Tix Mose lus feel bum aydly pupeselalr S. anatiiin war self Grvdinad) baditiny 1G5d, Cells of S. butantan (or S. anatum) were mixed with phage suspension, kept at room temperature for 30 to 60 minutes and plated out on agar plate. Many antigenic variant’ célonties .% were found the follow ng day. (Bh titer stock of phage is’so routine that it did not-even enter my head to record the above experiment. ) Title ; I am all for the title you have suggested. page 1 and 2, and page 16 section 12). I respect your opinion, Kindly do what you think would be best. page 18-19 15). You have suggested a shorter summary. I should think that it would not be often for non-member such as I to have his papers accepted. So I feel that I should take advantage of this opportunity and put in as much as I can, for I have no idea when my next chance will come. What is your opinion on this, Professor ? However, I have included a shorter summary in case you think I will be given another opportunity. Shorter summary of 15) : Taking H antigens beside 0 antigens into consideration, when O antigens of 8S, anatum, S.nyborg, S. Ot ekeri dis aeeteeete pe S. give and S. uganda are altered from 3, 10, to 3, 15, =n, these strains are changed to forms which are indtstingwish - ble from S. newington, S. selandia, Ss. cambridge, S..===as. new brunswick and S. kinshasa respectivély. On the other hand comparison of the sources from which they were isolated shows that &ach pair of S, anatum and §. newington, S. nyborg and S. selandia, S. give and Ss. new brunswick, and S, lexington and §. illinois has been £solated from similar sources respectively. Besides, the corresponding two types are identical or almost identical in biochemical behaviors with each other, These findings may suggest that type strains of E> group might have been yielded as a:restlt of tnfection of Ei groups organisms with phages, in nature, and this might be supported by the fact that more types have been isolated in Ej groupa than in E> group and each ty¥pélin Bp -gfowp == has its corresponding type in Ej group. FABLE £ Activity of bacteriophages obtained from Eo group organisms inducing changes in 0 antigens o Ey group organistis — PHAGES a Ht —t rN nN pO [M Ww fa fo o oO [h~ | + Eq GROUP STRAIN ala |= 1% é a Ia Ola 1S Ju jn ft. “1 [> Pp i]o |S jn [a fa fo ja wie | [se la jols ia Gis |Q fjajn jo ia fe wa fret | = 5 S&S |G ta{o Zz j]o |o “+ [@ ta ia wt 2 aia [9° | oO frei [> No. Type maja la lala lala ia 76 §. London 1416 -f-l. me S. give 316 --- +] + : § S. anatum 293 to] to} +o] o 0 o ° ° 1 S, amager 2399 lode 82 gs, zanzibar 5628 +fe]e 83 s, shangani 5630 ete-{- i101 Ss. uganda +1 + + + 189 s. butantan of ef of +o] tol +ol sof + 119 S. vejle +t + + - ~ Soh S. meleagridis +Jafoe> -[ =): 2 S. elisabethville t}al«ef -| -) 262 Ss, simi e*]fo*+f +7 -] + 139 S. weltevreden o+fel ef -] 4. 190 s, orion +] e} ele] +. 123 S. lexington *] oa} « 273 S. macallen tel + += Antigenic changes from 4,10 to 3,15 «= Antigenic changes from 4,10 to 3,10,15 -= No antigenic change within 6-10 subcultures +%-Standard strain number designated by F, Kauffmann °=Plaque formation TABLE 2 Changes in the 0 antigens of Ez group organisms, induced by bacteriophages obtained fron Eo group organisms n fe > ANTIGENIC Ez GROUP STRAIN 4 PHAGES R CHANGES fy o + . No.} © Type g From To 1] S. newington Co 1,3,19,19 [43,15 197; S. chittagong 1] S. canoga 1,3,10,19 14,15 a| mixture* 1,3,19 1,3,15,39 88} S. niloese 1236 | 11S. canoga S. senftenberg Aa! -~L{ S. canoga 3 3,15 iho| S: senftenberg- mixture * 1,3,19 1,3,15,19 simsbury l{ S. canoga S. senftenberg # USl = HS10 10} mixture Antigenic change No antigenic change within 6-10 subcultures Same as in fable Mixture of phages, and S$, ne brunswick, 1 8, obteined from S. newington, S. selandia and 1,19 antiserum, prepared by absorbing §. niloese "on antiserum witn S. londcn. TAPL& 3 Comparison of the sources between closely related pairs of types FIRST TYPH ISOLATKU FROM REPORT Animal Human 1919-20] S. anatum Ducklings (U.S.A,) Retail pork (U.S.A.) [Infantile diarrhoea (Uru. ) Chickens and turkeys (U.S.A.) Feces of healthy persons (Hune.3U.S.A.) Amer.sprav~dried eves (3.5.) Food poisoning (4%.2.) MLN of normal nies (U.S.A. 3éex. ) Gastroenteritis (U.S.A.) Silver fox (U.S.A.) Dog (U.S.A. ) et 1937 S,newineton Ducklings (U.S.A.) Retail pork (U.S.A.) | Gastroenteritis (U.S.A.) Chickens and turkeys (U.S.A.) Sewage (U.S.A) Amer,spray-dried ez (4.B.) Carriers (U.S.A.) NEN of normal pigs (U.S.A.;Uru.) Silver fox (U.S.A,) Dog (U.S.A.) 1936-37| S.nyborg a Gastroenteritis in a child (U.S.A.) 1937 5.selandia A young sailor with fever, lung symptoms and diarrhoea (Den. ) . 1937 S.give MLN of normal pigs (U.S.A.) Long-standing diarrhoea (Spain) Retail pork (U.S.A.) Chickens (U.S.A.) | Gastroenteritis:eateric tever (U.S.4A.) Gastroenteritis ot dog (U.S.A.) Carrier (U.S.A.) Amer. spray-dried eggs (%.5,) Dog(tiex.) : _ 1937 S.new brunswick] L:LN of normal nies (U.S.4.) Gastroenteritis in a woman (Den.) A baby chick (U.3.A,.) Dog (U.S.A. 3Mex.) | A patient who had returned from tropics (Den.) 1941 S,meleagridis |Turkey poults (U.S.A.) Typhoid-like tever (Venezuela;Medit. Area) MLN of normal pigs (Mex.) Intant diarrhoea (Uru.) Sewage" (U.S.A.) Reptiles (U.S.A.) Dog (Mex. ;U.S.A.) Gastroenteritis(U.S.A.) Carrier(Medit. drea) Amer. dried eggs (G.B.) Ge.suan soldier(Norway)Food poisoning(Medit. Area) 1947 S.cambridze A_soldier sutfering trom Sonne dysentery(G.B.) 1940 S, lexington Turkeys;MLN of normal nig (U.S.A.) Carrier (U.S.A.) 1941 S.illinois Turkeys (U.S.A.) Pigs (U.S.A.) Gastroenteritis(U.S.A.) Carrier(U.S.A.) Hungarian partridges (U.S.A.) 1940 S.uganda Pyrexia ot unknown origin(Uganda) 1950 S.kinshase LGh9 S.canoga 10-day old poult (U.S.A. ) ee 1930 S.senftenberg __-__|var,.neweastle _ Carrier (G.B.) _ 1929 S.senftenberg |Young turkeys(U.S.A.) Chickens(U.S.A.) | Gastroenteritis in a boy (Den. ) Chickens ers (U.S.A.:dapan;China) Gastroenteritis (U.S.A. ) Retail meat (U.S.A.) Carrier (U.S.A,) MLN of normal pies (Mex. ) _ 1942 S.simsbury Turkeys (U.S.A. ) Normal human teces(U.S.A.)Gastroenteritis(Medit. Area) MLN = Mesenterial lymph nodes Summarized trom: Rubin,H.L.et al. 1942 Arn. J.iya.,31,43-47;Hormaeche,Z.et 31.1943 Am, J.Diseases Children,46, 939-551; Edwards,P.R.et al.1943 J.Infectious Diseases,72,58~467;Cherry,W.B.et al.1943 Am.d.Hyg.,37,211-2153 Bruner,D.W.et al.1947 Am.J Hye. ,45,19-24;Woltf,A.M.et al.1948 Am.J.Public Health, 38,403-408;Wilson,%.S.et al. 1948 ;Breed,R.S.et al.1948;Felsenteld,O.et al.1951 éentr. Bakteriol. ,Parasitenk.Abt.I Ref. ,149, 351;Ball,MR. 1952 Zentr.Bakteriol. ,Parasitenk. Abt.I Ref. ,150,548;Varela,G.et al.1953 Zentr. Bakteriol. .Parasitenk, Abt.I Ref.,151,373;Gorham,J.R.et al.1953 Zentr. Bakteriol. ,Parasitenk. Abt. 1 Ref. ,151,373. 128 (Insert as indicated at page 12). Quantitative determinations of the inoidence of bacterial survivors and antigenic variants were made after thirty mimtes estposure to the phage (table 3). This experiment revealed the following: About one- twentieth of the treated cells survived. About half the surviving colonies formed non—transluoent colonies which were antigenic variants. A small number of translugcent colonies were also seen, all of these also variants. Studies are in progress to verify whether all lysogenised bacteria are antigenically altered, and vice versa (see section VI). IX. Effect of host strain on phage specificity. A small number of phage particles from an autolysate of S. canoga were propagated in series on S. anatum. After several passages, a suspension titratihg 4 x 10? per ml was obtained, This phage, which had been serially propagated on S. anatum was still effective in converting S. anatum and 3. butantan. Thus the effectiveness of the phage is independent of the propagating host (contra transduction, Zinder and Lederberg, 1952). 18A Many types showing O antigens 3,10 have been isolated which, with respech to their H antigenic complexes, are similar to corresponding 3,15 serotypes (e.g., 3. anatum- S, newington; S. nyborg- S. selandia). A consideration of the origins of these strains suggests that they have been found from similar sources. The corresponding pairs also tend to be sigilar in biochemicak behavior. It is therefore suggested that the experimental interconversions have been paralleled in nature {ard that each EB) serotype may be expected to have an Eg counterpart. Table 3 Survival and antigenic variation of S. anatum exposed to phage Dilution Colonies Translucent colonies Antigenic (average (all variant) variants from 5 (per 50 non- plates) translucent colonies tested) Active phage, 5x10°/m2 10 416 20 33 plus 8 S. anatum 10°/m1 1075 40 2 21 -b Heat—inactivated phage 10 104 0 0 plus 8 -7 a & anatum 10°/m1 lo 7 0 0 A phage suspension froa S. oanoga was mixed with cells of S. anatum (20 hour agar plate culture) at room teaperature for thirty minutes before plating. EKEEIAS 50 of each experiment mf aurviving colonies/were tested with 15 antiserum.