Bacteriophage Ecology Group
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© Stephen T. Abedon
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© Phage et al. last updated on Wednesday, May 17, 2000

THE ISOLATION OF T-EVEN PHAGES

(feces -?-> P.C./PC --> gamma --> T2; Sewage -?-> T4/T6)

Stephen T. Abedon, Department of Microbiology and Immunology
Arizona Health Sciences Center, University of Arizona, Tucson, AZ 85724
[T4 News (February 7)]

(A much modified version of this piece can be found in the
June, 2000, issue of the journal Genetics under the title
"The Murky Origin of Snow White and Her T-Even Dwarfs")

It may never prove possible to identify, with high confidence, the time, place, and source material for the original isolation of phages T2, T4, and T6. Nevertheless, a better understanding of where T-even phages came from has historical significance and is even of scientific consequence to those of us interested in the ecology of these organisms. Gathering clues for the discovery of a likely candidate for the original isolation of phage T2 was the more difficult task. For phages T4 and T6 this search was much less problematic and is presented first. I conclude with a conjecture on the history of free tryptophan (trp) adsorption cofactor dependence in these phages.

Evidence: Isolation of Phages T4 and T6 From Sewage

Demerec and Fano (1945) acknowledge the isolation of phages T4 and T6 (the "T" is for "type") as follows (absence of "T3" from the second half of quote is as published),

T3, T4, T5, and T6 were isolated from a mixture of phages supplied by Dr. Tony L. Rakieten . . . We are grateful to . . . (T.L.R.) of The Long Island College of Medicine, for a culture of mixed phages from which T4, T5, and T6 were isolated.

This wording suggests that phages T4 and T6 were isolated by Demerec and Fano (or, at least, not by Rakieten) from a single culture rather than received already purified. However, there is no information on from what, sewage versus feces, phages T4 and T6 were isolated. From what or where might Rakieten have obtained this culture?

The evidence suggests that T. L. Rakieten, and her frequent coauthor (and husband) M. C. Rakieten, considered sewage to be "an excellent source of bacteriophage of the coli-typhoid group" (Rakieten, 1932). Indeed, I could find no record of their isolation of coliphages from feces, but many instances of phage isolation from sewage (e.g., Rakieten & Rakieten, 1938; Rakieten et al., 1940; Rakieten & Bornstein, 1941; Rakieten & Rakieten, 1943). Thus, odds are that when Tony Rakieten supplied Demerec and Fano with "a mixture of phages," that mixture consisted either of raw sewage or, more likely (i.e., Rakieten et al., 1940), a phage lysate originally inoculated with raw sewage and grown, perhaps, using an Escherichia coli B host (1).

Evidence: Isolation of Phage T2 From Feces

Clues from the dawn of the classical period. Anderson presented a short history of phage T2 and E. coli B in a 1945 paper (1945a). He wrote (parentheses his, order of "Luria and Delbrück" is in error),

. . . Escherichia coli (strain PC of Kalmanson and Bronfenbrenner, '39, and called "B" by Luria and Delbrück, '42a). . . The virus T2 (called "PC phage" by Kalmanson and Bronfenbrenner, '39, and "gamma" by Luria and Delbrück, '42) . . .

Demerec and Fano's (1945) account of the origin of phage T2 (their reference representing the "final" naming of this phage) is consistent with Anderson's. Going to Delbrück and Luria (1942) confirms the change in the name of phage PC to gamma. However, they only, at best, ambiguously document a name change from E. coli PC to E. coli B. Importantly, I found no other corroboration for Anderson's (1945a) contention that E. coli B is derived from "B. coli PC." Nevertheless, Anderson clearly worked, as early as 1942, with both E. coli PC and "Bacteriophage anti-coli PC" (Luria & Anderson, 1942) (2). Unfortunately, Kalmanson and Bronfenbrenner (1939) do not say from where they obtained phage P.C. (3) or "Bacillus coli P.C. (4)" Nevertheless, there are some clues. For instance, they note that "B. coli P.C." was in use "over a period of 15 years" thus placing its isolation on or before 1924. Stated ambiguously, Delbrück (1946) notes that phage "PC" had "been used for many years in the laboratory of Prof. Bronfenbrenner."

Tracing phage PC back to 1927. A fortunate clue to the history of phage PC is presented by Kalmanson and Bronfenbrenner (1942). They note,

In studying the phenomenon of polyvalence, Bronfenbrenner (1933) employed a polyvalent phage (PC) which acted equally well on B. coli, B. dysenteriae Shiga, and B. dysenteriae Flexner . . . This page [sic] had been routinely carried on B. coli . . .

Bronfenbrenner (1933) notes that the (unfortunately unnamed in this 1933 study) phage employed is "one of the coli-phages in our collection, used in the various experiments in our laboratory for nearly 6 years and invariably found to be pure according to all accepted criteria . . ." That places phage PC in the Bronfenbrenner laboratory on or about February 1927, the same year that Bronfenbrenner describes unpublished experiments later repeated with phage P.C. (above).

The oldest reference I found to a phage PC was a 1932 paper (Bronfenbrenner, 1932) describing results obtained using "PC-coli and PC-shiga phages" which, presumably, infect "B. coli" and "B. shiga" respectively. This wording suggests that he is describing two distinct phages. However, Table I of this paper lists a "P.C. phage---B. coli" experiment plus a "P.C. phage---B. shiga" experiment. Thus, it is unclear whether phage PC is two distinct phages or just separate phage stocks distinguished by the host they were propagated on (e.g., Kalmanson & Bronfenbrenner, 1942).

Deciphering "P.C." Next I attempted to decipher the meaning of "P.C." For example, P.C. could be some (unknown) person's initials. Alternatively, P.C. might refer to a "Phage Coli" or "Polyvalent-phage Coli (5)." Another possibility is that P.C. stands for "pestis caviae." I explore this latter possibility below.

"Bacillus pestis caviae" is (or was) a mouse pathogen, causing a "mouse typhoid" (Bronfenbrenner & Korb, 1925d; Bronfenbrenner et al., 1926). This bacterium was used by Bronfenbrenner and others in some early phage studies (e.g., Bronfenbrenner & Korb, 1925c; Bronfenbrenner, 1927; Hetler & Bronfenbrenner, 1928). The evidence that "B. coli PC" is, or was derived from "B. pestis caviae" include: (i) the strain designation "P.C." is obviously derivable from the earlier species/subspecies designation "pestis caviae," (ii) "B. pestis caviae" was in use, as described by Bronfenbrenner et al. (1926), since at least 1922, (iii) "B. coli P.C." was in use since at least 1924 (above), (iv) I could find no reference to "B. coli P.C." before 1939, and (v) Bronfenbrenner et al. (1926; see also Bronfenbrenner & Korb, 1925d) isolated avirulent variants of "B. pestis caviae" (i.e., E. coli B is presumably avirulent).

To the contrary, there are incongruities to the assertion that the original host of phage PC was "B. pestis caviae." For instance: Why might the designation "B. pestis caviae" have been abandoned, seemingly without explanation, for "B. coli P.C.?" Why was a distinction between phages infecting "B. coli" and those infecting "B. pestis caviae" all but institutionalized by Bronfenbrenner (1928)? Why is "B. pestis caviae" not listed as a host for phage PC even when much is made of the polyvalent nature of this phage (i.e., Bronfenbrenner, 1933; Kalmanson & Bronfenbrenner, 1942)? Why, in one study in which "bacteriophage . . . coli (P.C.)" is employed (Bronfenbrenner & Sulkin, 1937), is a distinction still made between two phage types (i.e., "in the case of coli phage . . . in the case of the phage against B. pestis caviae")? Can E. coli B really be an avirulent derivative of a pathogenic bacterial strain? Thus, while "B. coli P.C." is "B. pestis caviae" may be a good idea, without better documentation it is questionable whether it is of significant utility.

Bronfenbrenner phage isolations documented. Since phage PC does not appear to have been referenced before the 1932 study mentioned above, the next logical step was to find a description by Bronfenbrenner (and others) for the acquisition or isolation of a coliphage having a "B. dysenteriae Shiga," "B. dysenteriae Flexner," "B. coli," or, even, "B. pestis caviae" host. For example, Bronfenbrenner & Korb (1925b) use several phages that appear to have been gotten from other laboratories. However, the 1925 date of this study suggests that is too early to account for the apparent 1927 acquisition of phage PC. Closer in time to this date Bronfenbrenner et al. (1926) noted that they had isolated "lytic filtrates yielded by the examination of over 300 mice" with most infecting "B. dysenteriae" and some infecting "B. pestis caviae."

Similarly, though much earlier, Bronfenbrenner & Korb (1924) describe phages "isolated October, 1923, from the stools of patients at the 'Babies' Hospital," one phage active against "B. dysenteriae Shiga," a second against "B. dysenteriae Flexner," and a third against "B. coli." They also noted that they later adapted what I guess from context to be the "B. coli" phage "to other strains of the colon dysentery group." In Bronfenbrenner and Korb (1925a) they describe the isolation of a phage active against "B. pestis caviae . . . from stools of mice surviving the experimental infection." Presumably they mean bacterial infection.

Significantly, I found no reference in a Bronfenbrenner study to the isolation of phages from sewage. Thus, Bronfenbrenner and his coworkers show a bias toward isolating phages from feces (see also Bronfenbrenner, 1928; Hershey & Bronfenbrenner, 1936). This tendency is not too surprising given the great influence of the fecal phage isolation precedent of d'Herelle (Bronfenbrenner, 1928; Adams, 1959). If phage PC was isolated by Bronfenbrenner (and this certainly is a possibility, perhaps a strong one), then it is likely that isolation was made from fecal material rather than from sewage. Unfortunately, the abysmal published record keeping of phage isolation by Bronfenbrenner (and others) will (if laboratory notebooks have been lost) likely keep the actual isolation of phage T2 forever shrouded in mystery.

Conclusion

To the extent that the evidence suggests that T4 and T6 came from a sample supplied by Tony Rakieten or from phages originally isolated by either Rakieten, I am comfortable concluding that phages T4 and T6 were originally isolated from sewage. In particular, it does not seem probable that phages T3, T4, T5, and T6 could have all been isolated from a single fecal sample supplied by a single individual. Nor is it likely that a phage worker would mix fecal samples from different individuals before setting out to isolate phages (though one never knows). I present the following passage as evidence that the two Rakietens took pride in the rigor with which they handled phage preparations. This statement also evidences just how difficult drawing usable information from pre-1940 phage literature can be (from Rakieten & Rakieten, 1938),

Since it is possible to maintain 'pure line' bacteriophage without significant observable changes over a period of years, the use of such phages in investigative work should be encouraged. The fact that bacteriophages, particularly those lysing organisms of the enteric group, are practically always mixtures of several types has made it difficult to duplicate the results of many published reports. Merely stating 'a coli phage was used' means little since there are undoubtedly hundreds of coli phages, each differing from the others by the combination of types they contain. Such an objection can be disregarded when identical phages are employed.

Similarly, to the extent that I am confident that phage PC was isolated in Bronfenbrenner's laboratory, I am comfortable concluding that the isolation was made from fecal material.

Trp dependence. The trp-dependence of T-even phages is likely a marker for colonic, as opposed to extracolonic propagation (Abedon, Ph.D. dissertation, U. of AZ, 1990). Yet, phage T4 and T6 are trp-dependent (Anderson, 1945b), a fact that questions my conclusion (above) that phages T4 and T6 were isolated from sewage. An obvious resolution of this apparent contradiction is that sewage is made up of fecal material, i.e., these phages were probably isolated one-step removed from the site of propagation of their ancestors.

More troubling is phage T2's lack of trp-dependence (Anderson, 1945b). This state of being has been described as exceptional among T-even phages (i.e., Kay & Fildes, 1963) and does not provide evidence (for or against) phage T2's presumptive (though perhaps questionable) isolation from feces. However, it is likely that phage PC in Bronfenbrenner's laboratory was propagated in synthetic medium lacking free tryptophan (Kalmanson & Bronfenbrenner, 1939; see also Hetler & Bronfenbrenner, 1928). Such growth conditions should readily select against trp-dependence (Delbrück, 1948). Thus, whatever the trp cofactor requiring status of the original T2 isolate, the laboratory propagation methods employed by Bronfenbrenner may have assured that the T2 isolate in use by the 1940s would not be trp-dependent. More to the point, we simply do not know whether the original T2 isolate was or was not trp-dependent.

Post Script

Anyone knowing of additional clues to the origins of phages T2, T4, and T6, or E. coli B, please send this information to myself or, for publication, to T4 News. Thank you to Bill Summers for his impressively informed "speculations and gossip."

End Notes

  1. Both Rakietens were post-doctoral fellows in d'Herelle's Yale laboratory between 1928 and 1933. They even inherited d'Herelle's Yale phage collection when he left (W.C. Summers, pers. commun.). Thus, through Tony Rakieten, T4 and T6 can claim "academic" descent from the laboratory of d'Herelle.

  2. Some have suggested that the "B" in E. coli B stands for Bordet. However, the apparent origin of E. coli B from Bronfenbrenner's laboratory naturally suggests that the "B" could stand for Bronfenbrenner (W.C.S., pers. commun.).

  3. Bronfenbrenner (and others) appear to have used the strain designations "P.C." and "PC" interchangeably (e.g., Kalmanson & Bronfenbrenner, 1939; Hershey et al., 1944).

  4. quoting passages I am using the convention of placing obsolete binomials in quotes.

  5. "phage coli-shiga".

Literature Cited

  1. Adams, M. H. 1959. Bacteriophages, Interscience, New York.

  2. Anderson, T. F. 1945a. On a bacteriolytic substance associated with a purified bacterial virus. J. Cell. Comp. Physiol. 25:1- 15.

  3. Anderson, T. F. 1945b. The role of tryptophane in the adsorption of two bacterial viruses on the host Escherichia coli. J. Cell. Comp. Physiol. 25:17-26.

  4. Bronfenbrenner, J. 1927. Studies on the bacteriophage of d'Herelle. VII. On the particulate nature of bacteriophage. J. Exp. Med. 45:873-886.

  5. Bronfenbrenner, J. 1928. Virus diseases of bacteria -- bacteriophagy, p. 373-414. In T. F. Rivers (ed.), Filterable Viruses. Williams & Wilkins, Baltimore, MD.

  6. Bronfenbrenner, J. 1932. The heat inactivation of bacteriophages. Proc. Soc. Exp. Biol. Med. 29:802-804.

  7. Bronfenbrenner, J. 1933. True polyvalence of pure bacteriophages. Proc. Soc. Exp. Biol. Med. 30:729-732.

  8. Bronfenbrenner, J., A. D. Hershey, and J. A. Doubly 1938. Evaluation of germicides by a manometric method. Proc. Soc. Exp. Biol. Med. 38:210-212.

  9. Bronfenbrenner, J., and C. Korb 1924. Effect of alcohol on the so-called bacteriophage of d'Herelle. Proc. Soc. Exp. Biol. Med. 21:177-179.

  10. Bronfenbrenner, J., and C. Korb 1925a. Studies on the bacteriophage of d'Herelle. II. Effect of alcohol on the bacteriophage of d'Herelle. J. Exp. Med. 42:419-429.

  11. Bronfenbrenner, J., and C. Korb 1925b. Studies on the bacteriophage of d'Herelle. III. Some of the factors determining the number and size of plaques of bacterial lysis on agar. J. Exp. Med. 42:483-497.

  12. Bronfenbrenner, J., and C. Korb 1925c. Studies on the bacteriophage of d'Herelle. IV. Concerning the oneness of the bacteriophage. J. Exp. Med. 42:821-828.

  13. Bronfenbrenner, J., and C. Korb 1925d. On variants of B. pestis caviae resistant to lysis by bacteriophage. Proc. Soc. Exp. Biol. Med. 23:3-5.

  14. Bronfenbrenner, J., R. S. Muckenfuss, and C. Korb 1926. Studies on the bacteriophage of d'Herelle. VI. On the virulence of the overgrowth in the lysed cultures of Bacillus pestis caviae (M. T. II). J. Exp. Med. 44:607-622.

  15. Bronfenbrenner, J., and S. E. Sulkin 1937. An attempt to purify bacteriophage by the procedure of Vinson. J. Infect. Dis. 61:259-263.

  16. Delbrück, M. 1946. Bacterial viruses or bacteriophages. Biol. Rev. 21:30-40.

  17. Delbrück, M. 1948. Biochemical mutants of bacterial viruses. J. Bacteriol. 56:1-16.

  18. Delbrück, M., and S. E. Luria 1942. Interference between bacterial viruses. I. Interference between two bacterial viruses acting upon the same host, and the mechanism of virus growth. Arch. Biochem. 1:111-141.

  19. Demerec, M., and U. Fano 1945. Bacteriophage-resistant mutants in Escherichia coli. Genetics 30:119-136.

  20. Hershey, A. D., and J. Bronfenbrenner 1936. Dissociation and lactase activity in slow lactose-fermenting bacteria of intestinal origin. J. Bacteriol. 31:453-464.

  21. Hershey, A. D., G. M. Kalmanson, and J. Bronfenbrenner 1944. Coordinate effects of electrolyte and antibody on the infectivity of bacteriophage. J. Immunol. 48:221-239.

  22. Hetler, D. M., and J. Bronfenbrenner 1928. Studies on the bacteriophage of d'Herelle. IX. Evidence of hydrolysis of bacterial protein during lysis. J. Exp. Med. 48:269-275.

  23. Kalmanson, G., and J. Bronfenbrenner 1939. Studies on the purification of bacteriophage. J. Gen. Physiol. 23:203-228.

  24. Kalmanson, G., and J. Bronfenbrenner 1942. Evidence of serological heterogeneity of polyvalent "pure line" bacteriophage. J. Immunol. 45:13-19.

  25. Kay, D., and P. Fildes 1962. The thermal lability of tryptophan-dependent bacteriophages. J. Gen. Microbiol. 27:147-153.

  26. Luria, S. E., and T. F. Anderson 1942. The identification and characterization of bacteriophages with electron microscope. Proc. Nat. Acad. Sci. U.S.A. 28:127-130.

  27. Rakieten, M. L. 1932. Studies with staphylococcus bacteriophage. I. The preparation of polyvalent staphylococcus bacteriophage. Yale J. Biol. Med. 4:807-818.

  28. Rakieten, M. L., and S. Bornstein 1941. Influence of certain bacteriophages on the H antigen of Salmonella poona and E. typhi. Proc. Soc. Exp. Biol. Med. 48:359-361.

  29. Rakieten, M. L., A. H. Eggerth, and T. L. Rakieten 1940. Studies with bacteriophages active against mucoid strains of bacteria. J. Bacteriol. 40:529-545.

  30. Rakieten, M. L., and T. L. Rakieten 1938. The inactivation of "pure line" phages by bacterial extracts and the loss of phage types in vivo. Yale J. Biol. Med. 10:191-208.

  31. Rakieten, T. L., and M. L. Rakieten 1943. Bacteriophagy in the developing chick embryo. J. Bacteriol. 45:477-484.

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