A fraction of otherwise antimicrobial-sensitive Bacillus subtilis cells, called persisters, are phenotypically tolerant of antimicrobial treatment. We report that, independently of B. subtilis' growth phase, transient ? toxin expression induces a dormant state and alters cellular responses so that cells are more sensitive to antimicrobials with different modes of action. This outcome is modulated by fine tuning (p)ppGpp and GTP levels: i) in the presence of low "dysregulated" (p)ppGpp levels (as in relA (-) cells) hyper-tolerance to both toxin and antimicrobials was observed; ii) physiological or low (p)ppGpp levels (as in the wild-type, sasA (-), sasB (-) and relA (-) sasA (-) context) show a normal toxin and antimicrobial tolerance; and iii) lower levels (in relA (-) sasB (-)) or absence of (p)ppGpp (in the relA (-) sasA (-) sasB (-) context), in concert with elevated GTP levels, potentiate the efficacy of both toxin and antimicrobial action, rendering tolerance vulnerable to eradication.
Bacterial nucleoid associated proteins play a variety of roles in genome maintenance and dynamics. Their involvement in genome packaging, DNA replication and transcription are well documented but it is still unclear whether they play any specific roles in genome repair. We discovered that untwisting of the DNA double helix by bacterial non-specific DNA binding proteins stimulates the activity of a repair endonuclease of the Nth/MutY family involved in abasic site removal during base excision repair. The essential Bacillus subtilis primosomal gene dnaD, coding for a protein with DNA-untwisting activity, is in the same operon with nth and the promoter activity of this operon is transiently stimulated by H(2)O(2). Consequently, dnaD mRNA levels persist high upon treatment with H(2)O(2) compared to the reduced mRNA levels of the other essential primosomal genes dnaB and dnaI, suggesting that DnaD may play an important role in DNA repair in addition to its essential role in replication initiation. Homologous Nth repair endonucleases are found in nearly all organisms, including humans. Our data have wider implications for DNA repair as they suggest that genome associated proteins that alter the superhelicity of the DNA indirectly facilitate base excision repair mediated by repair endonucleases of the Nth/MutY family.
Head-on encounters between the replication and transcription machineries on the lagging DNA strand can lead to replication fork arrest and genomic instability. To avoid head-on encounters, most genes, especially essential and highly transcribed genes, are encoded on the leading strand such that transcription and replication are co-directional. Virtually all bacteria have the highly expressed ribosomal RNA genes co-directional with replication. In bacteria, co-directional encounters seem inevitable because the rate of replication is about 10-20-fold greater than the rate of transcription. However, these encounters are generally thought to be benign. Biochemical analyses indicate that head-on encounters are more deleterious than co-directional encounters and that in both situations, replication resumes without the need for any auxiliary restart proteins, at least in vitro. Here we show that in vivo, co-directional transcription can disrupt replication, leading to the involvement of replication restart proteins. We found that highly transcribed rRNA genes are hotspots for co-directional conflicts between replication and transcription in rapidly growing Bacillus subtilis cells. We observed a transcription-dependent increase in association of the replicative helicase and replication restart proteins where head-on and co-directional conflicts occur. Our results indicate that there are co-directional conflicts between replication and transcription in vivo. Furthermore, in contrast to the findings in vitro, the replication restart machinery is involved in vivo in resolving potentially deleterious encounters due to head-on and co-directional conflicts. These conflicts probably occur in many organisms and at many chromosomal locations and help to explain the presence of important auxiliary proteins involved in replication restart and in helping to clear a path along the DNA for the replisome.
TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the trwC gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian cells.
Initiation of bacterial DNA replication at oriC is mediated by primosomal proteins that act cooperatively to melt an AT-rich region where the replicative helicase is loaded prior to the assembly of the replication fork. In Bacillus subtilis, the dnaD, dnaB and dnaI genes are essential for initiation of DNA replication. We established that their mRNAs are maintained in fast growing asynchronous cultures. DnaB is truncated at its C-terminus in a growth phase-dependent manner. Proteolysis is confined to cytosolic, not to membrane-associated DnaB, and affects oligomerization. Truncated DnaB is depleted at the oriC relative to the native protein. We propose that DNA-induced oligomerization is essential for its action at oriC and proteolysis regulates its localization at oriC. We show that DnaB has two separate ssDNA-binding sites one located within residues 1-300 and another between residues 365-428, and a dsDNA-binding site within residues 365-428. Tetramerization of DnaB is mediated within residues 1-300, and DNA-dependent oligomerization within residues 365-428. Finally, we show that association of DnaB with the oriC is asymmetric and extensive. It encompasses an area from the middle of dnaA to the end of yaaA that includes the AT-rich region melted during the initiation stage of DNA replication.
During DNA replication the helicase (DnaB) recruits the primase (DnaG) in the replisome to initiate the polymerization of new DNA strands. DnaB is attached to the tau subunit of the clamp-loader that loads the beta clamp and interconnects the core polymerases on the leading and lagging strands. The tau-DnaB-DnaG ternary complex is at the heart of the replisome and its function is likely to be modulated by a complex network of allosteric interactions. Using a stable ternary complex comprising the primase and helicase from Geobacillus stearothermophilus and the tau subunit of the clamp-loader from Bacillus subtilis we show that changes in the DnaB-tau interaction can stimulate allosterically primer synthesis by DnaG in vitro. The A550V tau mutant stimulates the primase activity more efficiently than the native protein. Truncation of the last 18 C-terminal residues of tau elicits a DnaG-stimulatory effect in vitro that appears to be suppressed in the native tau protein. Thus changes in the tau-DnaB interaction allosterically affect primer synthesis. Although these C-terminal residues of tau are not involved directly in the interaction with DnaB, they may act as a functional gateway for regulation of primer synthesis by tau-interacting components of the replisome through the tau-DnaB-DnaG pathway.
Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering.
The ?? module consists of a labile antitoxin protein, ?, which in dimer form (?(2)) interferes with the action of the long-living monomeric ? phosphotransferase toxin through protein complex formation. Toxin ?, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20-30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1-5×10(-5)). Early after induction ? toxin alters the expression of ?78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ? decreases synthesis of macromolecules and releases intracellular K(+). We propose that ? toxin induces reversible protective dormancy and permeation to PI, and expression of ?(2) antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (?10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ?(2) antitoxin, which blocks ATP binding by ? toxin, thereby inhibiting its phosphotransferase activity.
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