“ur Av YALE UNIVERSITY NEW HAVEN - CONNECTICUT DEPARTMENT OF BIOCHEMISTRY STERLING HALL OF MEDICINE 333 CEDAR STREET February 26, 1957 Dear Josh, Thank you for the reprints and, especially, for your note. I was delighted to find that someone remembers that strain SF work, and I do ap- preciate your comments. You may be amused to hear that only last Thursday I gave a report to our departmental lunch seminar on my old data "reconsidered" in the light of your Proceedings paper and Park's subsequent paper in Science. We also came to the conclusion that penicillin might be blocking the active uptake of glycine by the cells. However, the data might also mean that penicillin inhibits the mechanism of incorporating free glycine into cell wall peptide and that glycine,gin_ peptide alerady linkage gets incorporated by a penicillin-insensitive transfer. There isn't any way to decide this point at present. I have some data on the effect of penicillin on K12. It's not really "clean", and I've never considered it good enough to publish, but some of it may interest you. I gathered from your paper that you've had difficulty working with coli in synthetic medium, and I'm not surprised. Kl2 grows fairly well in the medium I used for strain SF (use either 5 g NaCl or 6.4 g KCl) with L-alanine (Semi 0.01 moles per liter) as the sole C= and Nesourcee The effects of penicillin, glycine, glycylglycine, and alanyl- alanine under these conditions are illustrated by the following table. Compound in moles/1 Ala (0.01) Ala + Cly Ala (.01) Alaala (0.005) (each 0,01) * + Glygly Gly (0.01) anne w een - ---- weneen------ (0,005)---------------------= Optical density reading at 30 hours (i.e., measure of lag time)when tubes contained at 0 hours 3x10? cells/ml. Control 0126 156 0161 172 + 10 Oxford units 266 -O71 ell) 2096 of pen. per ml. a + 20 Oxford units/ml! 2066 @009 2100 Oly oN Actually, the inhibition depends on the amount of gflycine added, the more glycine the more inhibition. I came to the conclusion, on the basis of viable cell counts, that the apparant inhibition by penicillin was due, in large part, to killing of cells. Moreover, if the reponse$to ala + glycine with and without 20 units of penicillin are compared, the kill due to penicillin occurs at just a about thetime when active cell multiplication starts in the absence of penicillin. From my experience with K12 and some of its mutants (58, 679), i#d say that the various strains may vary somewhat in their sensitivity to penicillin. K12 is always MORE sensitive to penicillin in that SF medium with alanine as C= and Ne source than it is in the presence of glucose and ammonium saltse How- ever, the concentration of inorganic salts mst be relatively high (just as I reported for strain SF). That salt effect is probably on the binding of penicil- YALE UNIVERSITY NEW HAVEN « CONNECTICUT DEPARTMENT OF BIOCHEMISTRY STERLING HALL OF MEDICINE 333 CEDAR STREET lin by the cells. For example, when strain SF is preincubated in SF minimal (devoid of C~ and Nesource) containing penicillin for 5 hours, and then transe ferred to fresh medium (no penicillin) with glycine - leucine, the cells show inhibited ability to grow. As you would expect there isn't any kill during the preincubation period. If the preincubation is carried out in SF minimal devoid of NaCl (or KCl), on transfer the cells behave as though they'd never been exposed to penicillin. I don't know if this is true of K1l2. By the way, the reason I asked you for the glycine auxotroph way back in 1952 was to use it for penicillin studies, Such studies have never been done = your auxotrophs turned out to be full of other interesting characteristice. I think I may have .ritten you that the serine auxotroph (1977) will grow on gly- cine peptides in the absence of serine, and, given a long enough incubation time, it even grows on free glycine. In fact, if the asparagine is omitted from Tatum's basal medium (or if glutamic acid is added to Tatum's medium) this so- called serine auxotroph responds quite well to free glycine, This is a pretty problem in glycine penetration all be itself, and it takes on added interest now in relation to the problem of penicillin effect. I hope to get back to this some day. Just now we are revising our book (poor fobls, wel), and I haven't done any lab work in menhbhs. Will you and Esther be going to the Federation meetings in Chicago in April? It's been much too long since we've seen you, and I hope Chicago is near enough to Madison for you to make the trips With best regards from Joey Sincerely, re P.Se Excuse this terrible typing. As W.M Clark once said, my typewriter doesn't know how to spell - and I don't give it much help.