Transcription initiation of the copy-number control and better-than-random segregation genes of the broad-host-range and low-copy-number plasmid pSM19035 are subjected to repression by the autoregulated pSM19035-encoded ω product in Bacillus subtilis cells. The promoters of the copS ( P cop1 and P cop2), δ ( P δ), and ω ( P ω) genes have been mapped. These promoters are embedded in a set of either seven copies of a 7-bp direct repeat or in a block consisting of two 7-bp direct repeats and one 7-bp inverted repeat; the blocks are present either two or three times. The cooperative binding of ω protein to the repeats on the P cop1, P cop2, P δ, and P ω promoters represses transcription initiation by a mechanism that does not exclude σ ^A RNAP from the promoters. These results indicate that ω protein regulates plasmid maintenance by controlling the copy number on the one hand and by regulating the amount of proteins required for better-than-random segregation on the other hand.

Cytological and genetic evidence suggests that the Bacillus subtilis DNA uptake machinery localizes at a single cell pole and takes up single-stranded (ss) DNA. The integration of homologous donor DNA into the recipient chromosome requires RecA, while plasmid establishment, which is independent of RecA, requires at least RecO and RecU. RecA and RecN colocalize at the polar DNA uptake machinery, from which RecA forms filamentous structures, termed threads, in the presence of chromosomal DNA. We show that the transformation of chromosomal and of plasmid DNA follows distinct pathways. In the absence of DNA, RecU accumulated at a single cell pole in competent cells, dependent on RecA. Upon addition of any kind of DNA, RecA formed highly dynamic thread structures, which rapidly grew and shrank, and RecU dissipated from the pole. RecO visibly accumulated at the cell pole only upon addition of plasmid DNA, and, to a lesser degree, of phage DNA, but not of chromosomal DNA. RecO accumulation was weakly influenced by RecN, but not by RecA. RecO annealed ssDNA complexed with SsbA in vitro, independent of any nucleotide cofactor. The DNA end-joining Ku protein was also found to play a role in viral and plasmid transformation. On the other hand, transfection with SPP1 phage DNA required functions from both chromosomal and plasmid transformation pathways. The findings show that competent bacterial cells possess a dynamic DNA recombination machinery that responds in a differential manner depending if entering DNA shows homology with recipient DNA or has self-annealing potential. Transformation with chromosomal DNA only requires RecA, which forms dynamic filamentous structures that may mediate homology search and DNA strand invasion. Establishment of circular plasmid DNA requires accumulation of RecO at the competence pole, most likely mediating single-strand annealing, and RecU, which possibly down-regulates RecA. Transfection with SPP1 viral DNA follows an intermediate route that contains functions from both chromosomal and plasmid transformation pathways. Many bacteria can actively acquire novel genetic material from their environment, which leads to the rapid spreading of, for example, antibiotic resistance genes. The bacterium Bacillus subtilis can differentiate into the state of competence, in which cells take up ssDNA through a DNA uptake complex that is specifically localized at a single cell pole. DNA can be integrated into the chromosome, via RecA, or can be reconstituted as circular dsDNA, if derived from plasmid or from viral DNA. We show that RecO, RecU, and Ku proteins, but not RecA, are important for plasmid transformation, and differentially accumulate at the polar DNA uptake machinery. Upon addition of any kind of DNA, the assembly of RecU at the competence pole dissipated, while RecA formed filamentous structures that rapidly grew and shrank within a 1 minute time scale. RecO visibly accumulated at the competence machinery only upon addition of plasmid DNA, but not of chromosomal DNA. In vitro, RecO was highly efficient at enhancing the annealing of complementary strands covered by SsbA, without the need for any nucleotide cofactor. The findings show that competent cells possess a dynamic recombination machinery and provide visual evidence for the existence of different pathways for transformation with chromosomal DNA or with plasmid DNA.

Bacillus subtilisRecU protein is involved in homologous recombination, DNA repair, and chromosome segregation. Purified RecU binds preferentially to three- and four-strand junctions when compared to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) (≈10- and ≈40-fold lower efficiency, respectively). RecU cleaves mobile four-way junctions but fails to cleave a linear dsDNA with a putative cognate site, a finding consistent with a similar genetic defect observed for genes classified within the ε epistatic group (namelyruvA,recD, andrecU). In the presence of Mg²⁺, RecU also anneals a circular ssDNA and a homologous linear dsDNA with a ssDNA tail and a linear ssDNA and a homologous supercoiled dsDNA substrate. These results suggest that RecU, which cleaves recombination intermediates with high specificity, might also help in their assembly.

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protects ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.