Journal of General Microbiology (19791, 115, 479-489. Printed in Great Britain 479 Indigenous Plasmids from Phytopathogenic Corynebacterium Species By D E N N I S C. GROSS, ANNE K. VIDAVER AND MICHAEL B. KERALIS Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68583, U.S.A. (Received 2 February 1979) Efficient and reproducible cell lysis and the isolation of plasmid DNA from nine phyto- pathogenic Corynebacterium species is described. One to three plasmids, ranging from 23 to 77 megadaltons, were recovered from each strain, as detected by comparative agarose gel electrophoresis. Corynebacteriummichiganense showed considerable plasmid diversity. Other species, such as C. nebraskense and C. insidiosum, did not. Bacteriocin production, colony morphology, pigmentation and virulence were not correlated with the presence of plasmids. Sedimentation through neutral and alkaline sucrose gradients and electrophoretic separation on agarose gels gave similar values for plasmid molecular weights. Log-linear alkaline sucrose gradients proved useful for molecular weight determinations and also for the isola- tion of purified plasmids on a preparative scale. INTRODUCTION The phytopathogenic Corynebacterium species are a diverse physiological, morphological and pathological group of Gram-positive bacteria that cause agronomically important diseases (Starr et al., 1975; Vidaver & Starr, 1979). Under natural conditions, Coryne- bacterium species are generally host-specific and cause diseases of distinct symptomatology (Lelliott, 1966). Serology (Lazar, 1968), phage sensitivity (Echandi & Sun, 1973) and bac- teriocin production (Gross & Vidaver, 1979) have all been used to differentiate species and strains of phytopathogenic corynebacteria. The ubiquity of bacteriocin production by 7 out of 12 phytopathogenic Corynebacterium species (Gross & Vidaver, 1979) and the general association of bacteriocin production by Gram-positive bacteria with plasmid DNA (Tagg et al., 1976) suggested a similar relationship may exist in corynebacteria. Indeed, strains of streptococci, clostridia and bacilli that lose plasmids often lose bacteriocin production (Tagg et al., 1976). However, there have been no reports of plasmids in Corynebacterium species including those of animal, plant or saprophytic origin. Currier & Nester (1976) reported a procedure for isolating both small and large [ > 100 megadalton (Mdal)] plasmids from Gram-negative bacteria. Plasmid enrichment involved denaturing sheared chromosomal DNA with alkali and selectively removing denatured DNA by salt precipitation in the presence of phenol. Chassy et al. (1976) used this method of plasmid isolation and developed a lysis procedure suitable for Gram-positive lactobacilli. Plasmid size determinations may be made by sedimentation through alkaline or neutral sucrose gradients (Nuti et al., 1977; Shipley & Olsen, 1975). However, Meyers et al. (1976) found agarose gel electrophoresis to be a rapid and economical means of surveying molecu- lar weights of plasmid DNA. All of the above techniques were adapted and used in this study. We report here a survey of strains from several phytopathogenic Corynebacterium species for the presence of plasmids. While the presence of plasmids and bacteriocin production were not associated, the number and molecular weight of plasmids detected revealed differ- ences in DNA content in otherwise indistinguishable strains. 0022-1287/79/0000-8627 $02.00 @ 1979 SGM