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,

No abstract

Bacillus pumilus ATCC 12140 harbors 10 or more copies per chromosome of each of two small plasmids. Variants of this strain were isolated which were sensitive to a killing activity produced by the plasmid-containing parent. Each of 24 such sensitive (S) variants tested lacked detectable levels of supercoiled deoxyribonucleic acid. Transduction of S variants to the Kill+ phenotype was performed using phage PBP1 propagated on a mutant of ATCC 12140, designated strain L10, that remained Kill+ but retained only a single plasmid species (plasmid pPL10; molecular weight,-4.4 x 106;-20 copies per chromosome; p = 1.698). Resulting Kill+ transductants of S variants contained a single plasmid species having a size and copy number comparable to that of pPL10. Transfer of pPL10 from strain L10 to B. pumilus strain NRS 576 was accomplished by transduction with selection for the Kill+ phenotype. Strain NRS 576 naturally harbors about two copies per chromosome of a 28-million-dalton plasmid, pPL576. In Kill+ transductants of NRS 576, plasmids pPL10 and pPL576 stably coexisted at a ratio of about 11 molecules of pPL10 to 1 molecule of pPL576. Therefore, pPL576 and pPL10 are compatible plasmids. B. subtilis 168 is naturally resistant to pPL10-determined killing activity. Plasmid pPL10 was therefore inserted into B. subtilis 168 by transformation, using an indirect selection procedure and a spoB mutant as recipient. The plasmid is stably maintained at an estimated 10 copies per chromosome in the spore-recipient and in spore+ transformants. pPL10 is sensitive to cleavage by the endonucleases Hind III and EcoRl.

A pseudolysogenic phage, PMB1, was isolated from soil on the basis of its ability to increase the sporulation frequency of the oligosporogenic Bacillus pumilus strain NRS 576 (sporulation frequency, <1%). Several spore-negative mutants (sporulation frequency, <10-8) derived from strain NRS 576, which were converted to spore positive by infection with PMB1, were subsequently identified. PMB1 repeatedly grown on a given spore-negative mutant (e.g., GW2) converted GW2 cells to spore positive. Each plaque-forming unit initiated the conversion of a spore-positive clone in semisolid agar overlays. GW2 cells remained spore positive as long as they maintained PMB1. Return of PMB1converted cells to the original spore-negative phenotype correlated with loss of PMB1. In liquid media, PMB1 infection increased the sporulation frequency of mutant GW2 over 106-fold. More than half of the spore-negative mutants we isolated from strain NRS 576 were converted to spore positive by PMB1 infection. PMB1-induced spores of the spore-negative mutant GW2 were somewhat more heat sensitive than uninfected or PMB1-infected spores of the sporepositive parent of GW2. PMB1-induced spores of GW2 do not differ from wildtype spores in morphology by phase-contrast microscopy, dipicolinic acid content, or rate of sedimentation through Renografin gradients.

The pseudotemperate phage PMB12 was isolated from soil on the basis of its ability to enhance the rate of sporulation of Bacillus subtilis 168. PMB12 was subsequently shown to convert the sporulation defect in two genetically distinct classes of sporulation mutants. One class includes those rifampin-resistant mutants that are also spore-negative (mutated at the rif locus). The other class includes a strain carrying the sporulation mutation spoCM-1. The spoCM-1 mutation is linked to cysA14 by PBS1 transduction but is distinct from the rif locus. Several other sporulation mutants were not converted by PMB12. PMB12 is related to phage PBS1. However, PBS1 did not convert the above sporulation mutants. The replication of PBS2, a clear-plaquing derivative of PBS1, is rifampin insensitive, apparently due to a phage-induced rifampin-insensitive RNA polymerase. PMB12 replication is also rifampin insensitive.