Cloning and characterization of a cluster of linked Bacillus subtilis late competence mutations

Abstract

We characterized a segment of chromosomal DNA from Bacillus subtilis that was required for the development of genetic competence. The chromosomal DNA was cloned from a group of genetically linked and phenotypically similar Tn9171ac insertion mutants deficient in competence. This cluster of mutations defined the comG locus. Chromosomal DNA flanking each of the six insertions was cloned. Restriction maps of the cloned plasmids revealed that their chromosomal inserts consisted of overlapping fragments. These data, together with Southern blots of chromosomal DNA from the comG mutants, showed that the six Tn9171ac comG insertions occurred in the following order: comG12, comG39, comG412, comG107, comG56, and comG210. Expression of the comG Tn9171ac insertions was from a promoter located upstream from the first insertion, comG12. This was determined genetically and by low-resolution S1 nuclease mapping of the 3' terminus. The comG region spanned about 5 kilobase pairs, based on low-resolution S1 nuclease mapping of the transcription terminator and Northern blotting. The comG12 mutation had a partial epistatic effect on the expression of one other com locus, comE, but none of the other comG mutations affected expression of this or any other com gene tested. Based on these conclusions, and on its size and phenotype, the comG locus must be organized as a polycistronic operon that is subject to competence-specific regulation.Genetic competence in Bacillus subtilis is an example of a regulated global response to environmental conditions and developmental signals. The final products of this regulated response are also of interest, because they are presumably required for the binding, processing, and transport of DNA from the medium into the cytosolic compartment. Little is known about these products or the specific roles they play (for a review, see reference 8), and it is of interest to characterize them on the molecular level. 30,32) provided us with powerful and convenient tools for cloning comnG DNA from B. subtilis. We were able to clone Tn9171ac insertion proximal chromosomal DNA from each of the six insertion mutants, obtaining in all about 6.5 kilobase pairs (kbp) of B. subtilis chromosomal DNA. We were also able to clone an intact region of comG DNA that was devoid of insertional disruptions and that consisted of about 8.5 kbp. We analyzed the cloned fragments by restriction mapping and determined the linear order of the six insertion positions in the comG region by Southern blotting. The epistatic relationships between comG and other competence genes were examined.(The work by M.A. was conducted in partial fulfillment of the requirements for the doctoral degree from the Department

The bdbDC Operon of Bacillus subtilisEncodes Thiol-disulfide Oxidoreductases Required for Competence Development

“…Among the very few examples of translocated proteins known to contain disulfide bonds in B. subtilis are ComGC (one intramolecular disulfide bond) and the ComGG homodimer (one intermolecular disulfide bond). These type IV pilin-like proteins form parts of the DNA uptake machinery and are required for DNA binding (5,33,34). Our experiments show that in the absence of either BdbD or BdbC, the Western blot signal for ComGC is markedly reduced.…”

Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein‐secretion apparatus: a general system for the formation of surface‐associated protein complexes

“…This phenotype is under the control of regulatory genes (Dubnau , 1991 ;Goodgal, 1982) and in B. subtilis genes necessary for competence are expressed late in the exponential phase of growth . Of the late competence loci characterized thus far there are two at which pil-like genes are found: comG, at which five genes have been identified (Albano et al , 1989;Breitling and Dubnau , 1990), and comG (Mohan et al, 1989). The first two comG genes express proteins homologous to PilB and PiIC, while the next three produce proteins with prepilin-like leader sequences (Fig .…”

Suppression of early competence mutations in Bacillus subtilis by mec mutations

“…The effects of these mutations were further investigated by testing their influences on expression of P-galactosidase from the comG12-lacZ fusion during growth in liquid CM ( (1,2). This fusion construct permitted the use of ,B-galactosidase for monitoring comG transcription.…”

“…The mutation in strain BD1512 was derived by replacing the Tn9171acZ insertion in comG12 by pTV21A2 (29), which confers chloramphenicol resistance (Cm'), cloning this chromosomal construct in Escherichia coli as a plasmid, and reintroducing the cloned construct into the B. subtilis chromosome by a Campbell-like recombination event (2). The resulting mutation is referred to as comG12(Campbelled).…”