Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110.

Abstract

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,

DNA-membrane association is necessary for initiation of chromosomal and plasmid replication in Bacillus subtilis.

“…Replication of pSLlO3 is via the pUBllO vector (18). The plasmid is 7.9 kilobases and can transform neomycin-sensitive B. subtilis to Nmr.…”

“…pSLlO3 is a chimera containing the Staphylocus aureus plasmid pUBIlO and an EcoRI restriction fragment from B. pumilus that can complement a defect in tryptophan biosynthesis (trpC) in B. subtilis (18). Replication of pSLlO3 is via the pUBllO vector (18).…”

“…B. subtilis 168 trp thy, 168 trp thy dna-i (16), and dnaBI9 thy (17) were used. The plasmid pSL103 (18) was introduced into these strains by first making the cells competent for transformation by the method of Bott and Wilson (19) and then transforming the strains with purified pSL103 DNA to neomycin resistance (Nmr). The cells were grown in SPC+ medium (20,21) supplemented with thymine at 5 ,ug/ml and required amino acids at 50 ,ug/ml.…”

GeneyerP, Involved in Surfactin Self-Resistance inBacillus subtilis

“…The SmaI-XbaI fragment of pUC18-yerP::Tn was blunt ended and ligated to the blunted HindIII site of pTB522, which autonomously replicates in B. subtilis (13). The ligation mixture was transformed into competent cells of the recA mutant MI112 to avoid integration into the recipient chromosome (16,40). The resulting pTB522-yerP::mini-Tn10 was transformed into B. subtilis strain 168.…”

In vitro type II binding of chromosomal DNA to membrane in Bacillus subtilis

“…chromosome and the plasmid pUB110 (10). Type II binding was found by an in vitro membrane-binding assay (3) using the chimeric plasmid pSL103 (2). Type II binding was found only in the pUB110 part of pSL103 and was localized to four distinct areas of the pUB110 molecule.…”