Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three-NCIB 3610 T , PY79, and SMY-we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.

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Toxin-antitoxin (TA) modules are pairs of genes in which one member encodes a toxin that is neutralized or whose synthesis is prevented by the action of the product of the second gene, an antitoxin, which is either protein or RNA. We now report the identification of a TA module in the chromosome of Bacillus subtilis in which the antitoxin is an antisense RNA. The antitoxin, which is called RatA (for RNA antitoxin A), is a small (222 nucleotides), untranslated RNA that blocks the accumulation of the mRNA for a toxic peptide TxpA (for toxic peptide A; formerly YqdB). The txpA and ratA genes are in convergent orientation and overlap by ca. 75 nucleotides, such that the 3 region of ratA is complementary to the 3 region of txpA. Deletion of ratA led to increased levels of txpA mRNA and lysis of the cells. Overexpression of txpA also caused cell lysis and death, a phenotype that was prevented by simultaneous overexpression of ratA. We propose that the ratA transcript is an antisense RNA that anneals to the 3 end of the txpA mRNA, thereby triggering its degradation.

The Streptococcusfaecalis transposon Tn917 was introduced into Bacillus subtilis by transformation of competent cells with the plasmid pAMal::Tn917 and was tested for transposition activity by selection for insertions into the temperate phage SPf. Insertions were obtained at a frequency indicating relatively efficient movement of the element, and Southern hybridization analysis of a particular insertion confirmed it to be the result of a genuine transposition event. A restriction fragment from pAMal::Tn917 containing the transposon sequences was ligated into a temperature-sensitive plasmid (pBD95), and transpositions into the B. subtilis chromosome were selected by requiring the transposon drug resistance to be maintained at temperatures nonpermissive for plasmid replication. Insertions have been recovered at many chromosomal sites, including ones that produced auxotrophy of different kinds and ones that produced various different sporulation-defective phenotypes, indicating good prospects for the use of Tn917 as a tool for insertional mutagenesis in B. subtilis.Tn elements (transposable drug resistance elements, transposons) have come into increasing prominence as important tools for genetic analysis, particularly in some of the Gram-negative species, such as Escherichia coli, in which indigenous elements have been extensively characterized and adapted for special applications (1). In species with less developed genetics, transposon manipulations clearly offer even greater advantages over existing possibilities for conventional genetic analysis and even greater improvements over available gene cloning methods, a fact that has motivated recent efforts to introduce some of the well-characterized enteric bacterial elements into such Gramnegative bacteria as Myxococcus (2) (7) and B. subtilis recE4 mutant strain BD224 (8) were obtained from D. Dubnau. B. subtilis strain CU1050 (9), which we used as lawn bacteria to visualize SP,3 phage plaques, and strain CU1147 (10), which we used as a source of SPJc2 phage, were obtained from S. A. Zahler. The Dedoner "kit" of mapping strains (11) was obtained from A. L. Sonenshein. All other strains were generated in this work and are described in the text.Culture Media. PAB is Bacto Penassay broth (Difco antibiotic medium no. 3); LB medium is Luria-Bertani medium, prepared according to Levine (12); DSM is Difco sporulation medium [Difco nutrient broth, with 0.5 ml of 1 M NaOH, 10 ml of 1.2% MgSO4, and 10 ml of 10% KCl per liter, and sup- Transformations and Transductions. Transformation-competent B. subtilis bacteria were prepared, stored, and transformed as described by . Standard methods were used for the preparation of PBS1 phage lysates and in performing transductions (15).Selection and Scoring of Genetic Markers. Chloramphenicol resistance (Cmr) was scored or selected on LB plates with Cm at 5 ,g/ml. The phenotype of resistance to macrolides, lincosamides, and streptogramin B antibiotics (MLSr) conferred by Tn917 is inducible by erythromycin (Em) (6). In LB broth,...

A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cmr) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cmr B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl2-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRI or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.