SINGLE-CELL ISOLATIONS OF DIPLOID HETEROZYGOUS ESCHERICHIA COLI M. R. ZELLE anp JOSHUA LEDERBERG Laboratory of Bacteriology, Cornell University, Ithaca, New York, and Department of Genetics, University of Wisconsin, Madison, Wisconsin Reprinted from JourRNAL or BacrERIOLOGY Vol. 61, No.3, March, 1951 Made in United States of America Reprinted from JOURNAL or BacreRIOLOGY Vol. 61, No. 3, March, 1951 SINGLE-CELL ISOLATIONS OF DIPLOID HETEROZYGOUS ESCHERICHIA COLI M.R. ZELLE ann JOSHUA LEDERBERG Laboratory of Bacteriology, Cornell University, Ithaca, New York, and Department of Gencties, University of Wisconsin, Madison, Wisconsin Received for publication December 28. 1959 In a previous report (Lederberg, 1949), certain cultures of Escherichia cola, strain Ix\-12, were described as heterozygous diploids. This conclusion was de- veloped from the following findings: (1) Unlike the parent cultures, or recombinants usually isolated by means of nutritional or drug-resistance selection from mixtures of complementary types, these exceptional cultures were extremely unstable. They repeatedly threw off new cultural types exhibiting various combinations of nutritional requirements and fermentative characteristics. The segregated types remained stable upon further subculture. (2) The presumed diploids were unstable only for characters in which the parents differed and were stable and similar to the parents for those in which the parents were alike. The characters studied included response to various bacteriophages as well as nutritional and fermentative qualities. (3) Diploid cultures selected from parents differing in several characters afforded the opportunity to study their association or linkage. When a segre- gated colony proved to be stable or pure for any single character, it was found to be pure for all. That is, segregation involves a complete block of differential characteristics. The instability is thus best understood as the oceasional separa- tion of blocks of characters derived from the two parents, but more or less per- manently associated. (4) The intracellular level of this association is a necessary condition of their description as heterozygous diploids. The arguments previously offered were probably valid, but mostly indirect and genetic. Aside from the fact that the unstable, segregating cultures could be maintained indefinitely through judi- ciously selected single-colony isolations, the segregants included a significant proportion of different, new combinations (see table 1). Recombination during segregation suggests an intracellular association of factors, unless extracellular hereditary influences are assumed. This last point is perhaps the least rigorous assertion of the previous work. The present investigation was undertaken to test this assertion by direct isola- tion cf single cells under microscopic observation. In work of this kind, a closely attached pair of cells might be mistaken for a single cell, which possibility re- quires the most critical exclusion, because of the cultural behavior of the pre- sumed diploids. Therefore, a method was adopted for following a pedigree from a single initial cell for several generations of fission. This technique has been used previously in a study of morphological variation in Salmonella (Zelle, 1942) and has been described in detail (Zelle, 1951). 351 352 M. R. ZELLE AND JOSHUA LEDERBERG Tvou. 61 TABLE 1 Fermentation tests on segregants isolated from individual Malv colonies 151 MAL-+ SELECTIONS 66 MAL— SELECTIONS No. Mal Mt | Xyl No. | Mal | wi | Xyi _|- ae __ 141 + + + 61 — _ | - 6 + - + 4 _ ; + - 3 + - | - ro | = | + + 1 + - + | Individual Mal» colonies were suspended in sterile waler and restreaked on maltose agar. No more than one Mal+ and one Mal— colony was obtained from an individual streaking. The larger number of tests on Mal+ is due in part to the preponderance of Mal-+ among segregants obtained in this way. Figure 1. Appearance of Malev colonies from a heterozygous diploid culture plated on eosin methylene-blue maltose agar. Pure Mal+ and Mal— colonies are also observed. 1951] DIPLOID HETEROZYGOUS ESCHERICHIA COLI 3038 EXPERIMENTAL RESULTS AND CONCLUSIONS Single-cell studies have been conducted on four different diploid cultures. The results obtained with one of these, H-226, will be recorded as typical. The parents of H-226 differ in the fermentation of maltose, xylose, and mannitol, as D=DIPLOID aan : S=SEGREGANT 1s Nea L=INVIABLE YY 7 _— D 7 N\ J x6 0 0 220 0 23 / NAT 0 tt oO tH 48 / LN “ee p 5 5 24 0 25 D \ NS / 26 D . 55 D 27 OL 27 i 13 “56 “28 D / _— B / WIP § 29 D s 29 20 $ \ > ep \ 4 Neocl2! § 14 307% 14 “122 S _l23 $ ez 4 \