Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells' genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.

• MeSH
• Bacillus subtilis cytology   genetics   metabolism   ultrastructure   MeSH
• DNA, Bacterial genetics   MeSH
• Conjugation, Genetic MeSH
• Microbial Viability * MeSH
• Nanotubes chemistry   MeSH
• Plasmids genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Bacterial MeSH

RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.

• Keywords
• RNAP, RNase J1, stalling, torpedo, transcription-replication collision,
• MeSH
• Bacillus subtilis enzymology   genetics   MeSH
• Bacterial Proteins metabolism   MeSH
• RNA, Bacterial genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases metabolism   MeSH
• Exoribonucleases metabolism   MeSH
• Transcription, Genetic MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• RNA, Bacterial MeSH
• DNA-Directed RNA Polymerases MeSH
• Exoribonucleases MeSH
• RNA, Messenger MeSH

The ybxF gene is a member of the streptomycin operon in a wide range of gram-positive bacteria. In Bacillus subtilis, it codes for a small basic protein (82 amino acids, pI 9.51) of unknown function. We demonstrate that, in B. subtilis, YbxF localizes to the ribosome, primarily to the 50S subunit, with dependence on growth phase. Based on three-dimensional structures of YbxF generated by homology modeling, we identified helix 2 as important for the interaction with the ribosome. Subsequent mutational analysis of helix 2 revealed Lys24 as crucial for the interaction. Neither the B. subtilis ybxF gene nor its paralogue, the ymxC gene, is essential, as shown by probing DeltaybxF, DeltaymxC, or DeltaybxF DeltaymxC double deletion strains in several functional assays.

• MeSH
• Bacillus subtilis genetics   growth & development   metabolism   MeSH
• Genes, Bacterial MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Gene Deletion MeSH
• Microscopy, Fluorescence MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Mutagenesis, Site-Directed MeSH
• Computer Simulation MeSH
• Recombinant Fusion Proteins chemistry   genetics   metabolism   MeSH
• Ribosomes metabolism   MeSH
• Protein Structure, Secondary MeSH
• Protein Binding MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Recombinant Fusion Proteins MeSH
• Green Fluorescent Proteins MeSH