Volume 1

Contemporary classics in the life sciences / edited by James T. Barrett.

Date:
[1986-]
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Contemporary classics in the life sciences / edited by James T. Barrett. Source: Wellcome Collection.

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    CC/NUMBER 19 _Thfs week's citation Classic : Spizizen I. Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. US 44 :1072-8, 1958. [Department of Microbiology, Western Reserve University School of Medicine, Cleveland, OHj In this paper, published 26 years ago, I dem onstrated DNA mediated genetic transfor mation in Bacillus subtilis As this organism could grow in simple minimal media, it was possible to utilize a variety of auxotrophic markers This made it possible to investigate the genetic controls of biosynthetic path ways as was being done in Escherichia coli using other gene transfer systems [The SC/® indicates that this paper has been cited in over 1,010 publications since 1958 ] John Spizizen Department of Microbiology and Immunology University of Arizona Health Sciences Center Tucson, AZ 85724 February 27, 1984 We had previously attempted to trans form Escherichia coli auxotrophs without success. However, DNA-protein complexes prepared from denatured T2 bacteriophage were found to 'transform' £ coli proto plasts, 1 that is, replication and maturation of the phage would occur. These experiments suggested to us that cell wall components were barriers to naked DNA entry. It was at this time that I speculated that Bacillus sub tilis might be a promising organism, since during stages of outgrowth of germinating spores, cell wall components were minimal or entirely absent. Fortunately, Charles Yanofsky was able to provide me with several stable auxotro phic mutants of B subtilis which had been isolated by Burkholder and Giles at Yale University. 2 Transformation of an indole-re- quiring strain (168) with DNA isolated from a wild strain (W23) was readily achieved. Although germinating spores were initially employed, vegetative cells grown in minimal medium suitably supplemented with a low concentration of casein hydrolysate were found to be highly transformable. Refine ments to achieve optimal conditions for transformation were later introduced. 3 These studies demonstrated that specific growth conditions presumably affecting wall synthesis would allow cells to become 'com petent' for transformation. The original con cept that spore germination would provide the competent state was thus incorrect. Fur thermore, other processes besides DNA per meability are now known to be involved in DNA transformation by competent cells. These include a series of coordinated enzy matic steps following DNA attachment, DNA cutting, separation of double strands, penetration of single strands, and pairing with homologous regions of the resident chromosome. The specific enzymes and genetic control of these reactions remain to be identified. B subtilis strain 168 has the capability of carrying out this coordinated process. It was fortuitous that this strain was used as many if not most strains, including the donor strain W23, could not be made competent for chromosomal DNA transfor mation. Many papers describing modified proce dures for transformation have appeared. However, continued reference to the origi nal publication has been made mainly be cause of the minimal medium used. This sim ple medium has been employed for transfor mation in numerous laboratories, as well as for bacterial growth when supplemented with casein hydrolysate and required growth factors. It is ironic that this minor item in the publication is the one quoted most frequent ly Nevertheless, this paper provided im petus for investigations of new areas of pro- caryotic genetics. These include the genetic control of sporulation, a primitive form of differentiation, and synthesis of extracellu lar proteins and wall polymers characteristic of gram-positive bacteria. 4 I was fortunate to have been stimulated by Yanofsky and Howard Gest to initiate and pursue these studies while at Western Re serve University. 1 Spizizen I. Infection of protoplasts by disrupted T2 virus. Proc Nat Acad Sci US 43:694-701. 1957. 2 Burkholder P R & Giles N H, Jr. Induced biochemical mutations in Bacillus subtilis. Amer J Bot 34:345-8. 1947. (Cited 105 times since 1955.) J Anagnostopoulos C & Spizizen I. Requirements for transformation in Bacillus subtilis J Bacteriology 81:741-6, 1961. (Cited 615 times.) 4. Dubnau D A, ed. The molecular biology of the bacilli. Volume I. Bacillus subtilis. New York: Academic Press. 1982 378 p.
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