A conserved anti-repressor controls horizontal gene transfer by proteolysis

The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in the Bacillus subtilis chromosome. The SOS response and the RapI-PhrI sensory system activate ICEBs1 gene expression, excision and transfer by inactivating the ICEBs1 repressor protein ImmR. Although ImmR is similar to many characterized phage repressors, we found that, unlike these repressors, inactivation of ImmR requires an ICEBs1-encoded anti-repressor ImmA (YdcM). ImmA was needed for the degradation of ImmR in B. subtilis. Coexpression of ImmA and ImmR in Escherichia coli or co-incubation of purified ImmA and ImmR resulted in site-specific cleavage of ImmR. Homologues of immR and immA are found in many mobile genetic elements. We found that the ImmA homologue encoded by B. subtilis phage phi105 is required for inactivation of the phi105 repressor (an ImmR homologue). ImmA-dependent proteolysis of ImmR repressors may be a conserved mechanism for regulating horizontal gene transfer.     

Regulation of horizontal gene transfer in Bacillus subtilis by activation of a conserved site-specific protease

The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in Bacillus subtilis. The RecA-dependent SOS response and the RapI-PhrI cell sensory system activate ICEBs1 gene expression by stimulating cleavage of ImmR, the ICEBs1 immunity repressor, by the protease ImmA. We found that increasing the amount of wild-type ImmA in vivo caused partial derepression of ICEBs1 gene expression. However, during RapI-mediated derepression of ICEBs1 gene expression, ImmA levels did not detectably increase, indicating that RapI likely activates the protease ImmA by increasing its specific activity. We also isolated and characterized mutations in immA (immA(h)) that cause partial derepression of ICEBs1 gene expression in the absence of inducing signals. We obtained two types of immA(h) mutations: one type caused increased amounts of the mutant proteins in vivo but no detectable effect on specific activity in vitro; the other type had no detectable effect on the amount of the mutant protein in vivo but caused increased specific activity of the protein (as measured in vitro). Together, these findings indicate that derepression of ICEBs1 gene expression is likely caused by an increase in the specific activity of ImmA. Homologs of ImmA and ImmR are found in many mobile genetic elements, so the mechanisms that regulate ImmA-mediated cleavage of ImmR may be widely conserved.     

Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are mobile genetic elements that can transfer from one bacterial cell to another by conjugation. ICEBs1 is integrated into the trnS-leu2 gene of Bacillus subtilis and is regulated by the SOS response and the RapI-PhrI cell-cell peptide signaling system. When B. subtilis senses DNA damage or high concentrations of potential mating partners that lack the element, ICEBs1 excises from the chromosome and can transfer to recipients. Bacterial conjugation usually requires a DNA relaxase that nicks an origin of transfer (oriT) on the conjugative element and initiates the 5'-to-3' transfer of one strand of the element into recipient cells. The ICEBs1 ydcR (nicK) gene product is homologous to the pT181 family of plasmid DNA relaxases. We found that transfer of ICEBs1 requires nicK and identified a cis-acting oriT that is also required for transfer. Expression of nicK leads to nicking of ICEBs1 between a GC-rich inverted repeat in oriT, and NicK was the only ICEBs1 gene product needed for nicking. NicK likely mediates conjugation of ICEBs1 by nicking at oriT and facilitating the translocation of a single strand of ICEBs1 DNA through a transmembrane conjugation pore.     

The Bacillus subtilis conjugative transposon ICEBs1 mobilizes plasmids lacking dedicated mobilization functions

Integrative and conjugative elements (ICEs, also known as conjugative transposons) are mobile elements that are found integrated in a host genome and can excise and transfer to recipient cells via conjugation. ICEs and conjugative plasmids are found in many bacteria and are important agents of horizontal gene transfer and microbial evolution. Conjugative elements are capable of self-transfer and also capable of mobilizing other DNA elements that are not able to self-transfer. Plasmids that can be mobilized by conjugative elements are generally thought to contain an origin of transfer (oriT), from which mobilization initiates, and to encode a mobilization protein (Mob, a relaxase) that nicks a site in oriT and covalently attaches to the DNA to be transferred. Plasmids that do not have both an oriT and a cognate mob are thought to be nonmobilizable. We found that Bacillus subtilis carrying the integrative and conjugative element ICEBs1 can transfer three different plasmids to recipient bacteria at high frequencies. Strikingly, these plasmids do not have dedicated mobilization-oriT functions. Plasmid mobilization required conjugation proteins of ICEBs1, including the putative coupling protein. In contrast, plasmid mobilization did not require the ICEBs1 conjugative relaxase or cotransfer of ICEBs1, indicating that the putative coupling protein likely interacts with the plasmid replicative relaxase and directly targets the plasmid DNA to the ICEBs1 conjugation apparatus. These results blur the current categorization of mobilizable and nonmobilizable plasmids and indicate that conjugative elements play a role in horizontal gene transfer even more significant than previously recognized.     

Synethesis and integration of viral DNA in chicken cells at different time after infection with various multiplicities of avian oncornavirus.

To see if integration of the provirus resulting from RNA tumor virus infection is limited to specific sites in the cell DNA, the variation in the number of copies of virus-specific DNA produced and integrated in chicken embryo fibroblasts after RAV-2 infection with different multiplicities has been determined at short times, long times, and several transfers after infection. The number of copies of viral DNA in cells was determined by initial hybridization kinetics of single-stranded viral complementary DNA with a moderate excess of cell DNA. The approach took into account the different sizes of cell DNA and complementary DNA in the hybridization mixture. It was found that uninfected chicken embryo fibroblasts have approximately seven copies, part haploid genome of DNA sequences homologous to part of the Rous-association virus 2 (RAV-2) genome. Infection with RAV-2 adds additional copies, and different sequences, of RAV -2- specific DNA. By 13 h postinfection, there are 3 to 10 additional copies per haploid genome. This number can not be increased by increasing the multiplicity of infection, and stays relatively constant up to 20 h postinfection, when some of the additional viral DNA is integrated. Between 20 and 40 h postinfection, the cells accumulated up to 100 copies per haploid genome of viral DNA. Most of these are unintegrated. This number decreases with cell transfer, until cells are left with one to three copies of additional viral DNA sequences per haploid genome, of which most are integrated. The finding that viral infection causes the permanent addition of one to three copies of integrated viral DNA, despite the cells being confronted with up to 100 copies per haploid genome after infection, is consistent with a hypothesis that chicken cells contain a limited number of specific integration sites for the oncornavirus genome.