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The chloramphenicol (Cm)-inducible emL4 gene of Tn1696 specifies nonenzymatic resistance to Cm and is regulated by attenuation.

Approximately 3% of the deoxyribonucleic acid (DNA) of Bacillus pumilus NRS576 can be isolated as covalently closed, circular duplex molecules of homogeneous size. The mol wt of the 576 plasmid is approximately 30 million. NRS576 (plasmid+) is oligosporogenic; less than 1% of the cells form spores during incubation in liquid AK sporulation medium. Variants that form spores at a high frequency (greater than 50% spores in 24 h) occur spontaneously at a frequency of approximately 10'. More than 25 such variants have been examined and all lack detectable plasmid DNA. A relationship appears to exist between the oligosporogenic properties of NRS576 and the presence of the 576 plasmid.The existence of extrachromosomal genetic determinants (plasmids) is well documented for members of the Enterobacteriaceae, Staphylococcus aureus, and species of Pseudomonas (4,8,9,18). Those plasmids which have been mo §t extensively studied are characteristically small, covalently closed, circular duplex deoxyribonucleic acid (DNA) molecules which confer on the host known genetic functions. Examples of plasmid-directed host properties include the ability to conjugate, resistance to antibiotics and/or inorganic ions, and the production of colicins (18). Although most known plasmids were initially detected because of their genetic influence on the host, the development of biochemical techniques which permit the isolation of small, circular, duplex DNA molecules (21) has allowed the demonstration of plasmid-like mini-circle DNA in the absence of known genetic functions for the presumed extrachromosomal elements (5,12,19,23).Evidence for naturally occurring plasmids in sporeforming bacteria is presently limited to a study of the plasmid-like minicircular DNA isolated from Bacillus megaterium (3). Approximately 30 to 40% of the DNA of a strain of B. megaterium can be isolated as small, circular duplex molecules of sizes ranging from 0.8 to 34 ,um. These heterogeneous minicircles share extensive nucleotide sequence homology with the chromosomal DNA (Henneberry and Carlton, 1971, Bacteriol. Proc., p. 45; manuscript in preparation), and no genetic function has been attributed to them. It is presently unclear whether any of these heterogeneous minicircles are functionally analogous to plasmids, or whether they represent degraded and circularized chromosomal DNA.In the present report, evidence is presented which demonstrates that approximately 3% of the DNA of a strain of B. pumilus (a species related to B. subtilis [15][16][17]24]) can be isolated as circular duplex molecules of homogeneous size. Spontaneous variants that exhibit an increased frequency of sporulation lack detectable plasmid DNA.MATERIALS AND METHODS Bacteria. B. pumilus NRS576, obtained from R. E. Gordon, is sensitive to the transducing phage PBP1 (13) and does not exhibit the high rate of spontaneous mutation that is characteristic of a previously described strain of B. pumilus (14). In several experiments, two nitrosoguanidine-induced (16) auxotrophic derivatives of NRS576 were...

ABSTRACr Plasmid pUB10 (-2.8 (1,2). Direct application of recombinant DNA technology to the study of Bacillus subtilis will ultimately provide a general method for constructing partial diploid strains which will, in turn, permit genetic complementation analyses of specific mutations and provide a source of easily obtainable DNA highly enriched for genes of chromosomal origin whose in vitro expression may be of special interest such as sporulation genes.Among those Bacillus plasmids currently available, three determine host functions that do not permit direct selection of plasmid-containing transformants of B. subtilis (3-5), or the plasmids govern no known host function [i.e., they are cryptic plasmids (6-8)]. In contrast, certain antibiotic resistance plasmids originally detected in Staphylococcus aureus have recently been transformed into B. subtilis where they are stably maintained and express the appropriate antibiotic resistance trait (ref. 9). In the present report we describe the properties of one such antibiotic resistance plasmid, pUB110, and the use of this plasmid for cloning EcoRl endonuclease-generated DNA fragments in B. subtilis 168 (e.g., 1 jsg), as monitored by agarose gel electrophoresis, was routinely used to digest the plasmid. The same ratio of enzyme activity to DNA concentration was used to digest phenol-purified cell DNA extracted from B. licheniformis strains 9945A and 749C, B. pumilus NRRL B-3275, and B. pumilus NRS 576 (Table-1). After digestion, enzyme activity was terminated by incubating the DNA-containing solutions at 65°for 15 min. Annealing of the cohesive ends was achieved by combining digested cellular DNA (3 gg) with EcoRl-cleaved pUB110 (0.5 ,ug), both in EcoRl digestion buffer (13), and holding the mixture at 20 for 18 hr. The solution (generally 100-200 1l) was adjusted by adding dithiothreitol to 10 mM, ATP to 50 mM, and 1 unit of T4-induced DNA ligase (Miles Laboratories). Incubation was then continued for 8 hr at 100 and then 8 hr at 150. The resulting DNA preparations were dialyzed against 2-f[Tris(hydroxymethyl)methyl]aminolethanesulfonate (TES) buffer (3) prior to use.Ligated DNA preparations (2 ,ug) were added to 5 X 108 competent (17) BRI51 cells (trpC2 metBlO lys-3). Cells and DNA were shaken for 1 hr at 370 and the entire 1-ml transformation mixture was diluted into 20 ml of PB containing neomycin sulfate (5 ,gg/ml). Controls, including cells not exposed to DNA and DNA incubated without cells, were similarly treated. After overnight incubation at 370, only the culture containing the transformed cells had grown to saturation. Portions of this culture were washed with Spizizen minimal medium and diluted 1:10 into several 10-ml portions of Spizizen minimal medium supplemented with 0.05% acid-hydrolyzed casein and neomycin sulfate. Each of these cultures was then shaken at 370 overnight, at which time each had grown to saturation. A loopful of each culture was streaked to minimal agar containing 0.5% casein hydrolysate. After overnight incubation,

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