Tetracycline, an Appropriate Reagent for Measuring Bone-Formation Activity in the Murine Model of the Streptococcus mutans-Induced Bone Loss

Tetracycline is used as a fluorescent reagent to measure bone formation activity in bone histomorphometric analyses. However, there is a possibility to lead a different conclusion when it is used in a bacteria-infected murine model since the tetracycline is considered to work as an antibiotic reagent. There are non-antibiotic fluorescent reagents such as alizarin and calcein for measuring bone formation activity. The purpose of this study was to clarify whether tetracycline could be an appropriate reagent to measure bone formation activity in a murine bacterial model in the same way as a non-antibiotic fluorescent reagent. We used Streptococcus mutans (S. mutans), a normal inhabitant in the oral cavity and tetracycline-sensitive bacteria, for inducing the bacterial model. The murine bacterial model was generated by intravenously inoculating S. mutans to the tail vein, followed immediately by the injection of the first fluorescent reagent, and the second one was injected 2 days prior to euthanization. After one day of inoculation with S. mutans, the subcutaneously injected alizarin had a similar colony count derived from the liver and the bone marrow tissue compared to the phosphate buffered saline (PBS)-injected control group. On the other hand, subcutaneous injection of tetracycline led to a significantly lower colony count from the liver compared to alizarin- or calcein-injected group. However, on day seven, after S. mutans intravenous injections, bone mineral density of distal femurs was significantly reduced by the bacteria inoculation regardless of which fluorescent reagents were injected subcutaneously. Finally, S. mutans inoculation reduced bone-formation-activity indices in both the tetracycline-alizarin double-injected mice and the calcein-alizarin double-injected mice. These results suggested that a one-time injection of tetracycline did not affect bone formation indices in the S. mutans-induced bone loss model. Tetracycline could be used for measuring bone formation activity in the same way as non-antibiotic fluorescent reagent such as calcein and alizarin, even in a tetracycline-sensitive bacterium-infected model.

Identifying small ORFs in prokaryotes with ribosome profiling

Small proteins encoded by ORFs shorter than 50 codons (sORFs) are often overlooked by annotation engines and are difficult to characterize using traditional biochemical techniques. Ribosome profiling has tremendous potential to empirically improve the annotations of prokaryotic genomes. Recent improvements in ribosome profiling methods for bacterial model organisms have revealed many new sORFs in well-characterized genomes. Antibiotics that trap ribosomes just after initiation have played a key role in these developments by allowing unambiguous identification of the start codons (and hence the reading frame) for novel ORFs. Here we describe these new methods and highlight critical controls and considerations for adapting ribosome profiling to different prokaryotic species.

Nanopore sequencing of RNA and cDNA molecules expands the transcriptomic toolbox in prokaryotes

High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely employed to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism Escherichia coli, which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that cDNA-seq offers improved yield and accuracy without bias in quantification compared to direct RNA sequencing. Notably, cDNA-seq can be readily used for simultaneous transcript quantification, accurate detection of transcript 5 ′ and 3′ boundaries, analysis of transcriptional units and transcriptional heterogeneity. In summary, we establish Nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features thereby advancing it to become a standard method for RNA analysis in prokaryotes.

TatA and TatB generate a hydrophobic mismatch that is important for function and assembly of the Tat translocon in Escherichia coli

The Tat system translocates folded proteins across energy-transducing prokaryotic membranes. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was tolerated. Length-variation in TatB clearly destabilized TatBC-containing complexes, indicating that the 12-residues-length is crucial for Tat component interactions and translocon assembly. Metal tagging transmission electron microscopy revealed the dimensions of TatA assemblies, which prompted molecular dynamics simulations. These showed that interacting TMHs of larger TatA assemblies can thin the membrane together with laterally aligned tilted APHs that generate a deep V-shaped groove. The conserved hydrophobic mismatch may thus be important for membrane destabilization during Tat transport, and the exact length of 12 hydrophobic residues could be a compromise between functionality and proton leakage minimization.

Study of the solubilisation process of bacterial model membranes induced by DDAO

Solubilisation of two bacterial model membranes induced by N,N-dimethyl-1-dodecanamine-N-oxide (DDAO) was studied. The first model membrane consisted of a mixture of palmitoyloleoylphosphatidylethanolamine (POPE) and palmitoyloleoylphosphatidylglycerol (POPG) in a molar ratio 0.6:0.4 mol/mol, and a second model membrane was enriched with tetraoleoylcardiolipin (TOCL) with a composition POPE-POPG-TOCL = 0.67:0.23:0.1 mol/mol/mol. Solubilisation of these model membranes was studied by static light scattering (nephelometry). Effective ratio Re (the amount of DDAO integrated into the bilayer to the amount of lipid) at different steps of the solubilisation process was determined. The molar partition coefficient of DDAO was calculated – in case of the POPE-POPG membrane, Kp = 5,300 ± 400, for the POPE-POPG-TOCL membrane, Kp = 6,500 ± 500.

A Cell-Free Screen for Bacterial Membrane Disruptors Identifies Mefloquine as a Novel Antibiotic Adjuvant

Antibacterial discovery efforts have lagged far behind the need for new antibiotics. An approach that has gained popularity recently is targeting bacterial phospholipid membranes. We leveraged the differences between bacterial and mammalian phospholipid compositions to develop a high-throughput screen that identifies agents that selectively disrupt bacterial membranes while leaving mammalian membranes intact. This approach was used to screen 4480 compounds representing a subset of the Maybridge HitFinderTM V.11 Collection and the Prestwick Chemical Drug Library®. The screen identified 35 “positives” (0.8% hit rate) that preferentially damage bacterial model membranes. Among these, an antimalarial compound, mefloquine, and an aminoglycoside, neomycin, were identified. Further investigation of mefloquine’s activity against Staphylococcus aureus showed that it has little antibiotic activity on its own but can alter membrane fluidity, thereby potentiating a β-lactam antibiotic, oxacillin, against both methicillin-susceptible and methicillin-resistant S. aureus. This study indicates that our cell-free screening approach is a promising platform for discovering bacterial membrane disruptors as antibacterials antibiotic adjuvants.

Asymmetric chromosome segregation and cell division in DNA damage-induced bacterial filaments

The DNA damage response and cell division checkpoints have been well studied in several bacterial model systems, but how cells exit such a checkpoint to restart wild-type growth is unclear. This study highlights a central function for asymmetric division in mediating cellular recovery from DNA damage.