We previously reported that lagging-strand genes accumulate mutations faster than those encoded on the leading strand in Bacillus subtilis . Although we proposed that orientation-specific encounters ...between replication and transcription underlie this phenomenon, the mechanism leading to the increased mutagenesis of lagging-strand genes remained unknown. Here, we report that the transcription-dependent and orientation-specific differences in mutation rates of genes require the B. subtilis Y-family polymerase, PolY1 ( yqjH ). We find that without PolY1, association of the replicative helicase, DnaC, and the recombination protein, RecA, with lagging-strand genes increases in a transcription-dependent manner. These data suggest that PolY1 promotes efficient replisome progression through lagging-strand genes, thereby reducing potentially detrimental breaks and single-stranded DNA at these loci. Y-family polymerases can alleviate potential obstacles to replisome progression by facilitating DNA lesion bypass, extension of D-loops, or excision repair. We find that the nucleotide excision repair (NER) proteins UvrA, UvrB, and UvrC, but not RecA, are required for transcription-dependent asymmetry in mutation rates of genes in the two orientations. Furthermore, we find that the transcription-coupling repair factor Mfd functions in the same pathway as PolY1 and is also required for increased mutagenesis of lagging-strand genes. Experimental and SNP analyses of B. subtilis genomes show mutational footprints consistent with these findings. We propose that the interplay between replication and transcription increases lesion susceptibility of, specifically, lagging-strand genes, activating an Mfd-dependent error-prone NER mechanism. We propose that this process, at least partially, underlies the accelerated evolution of lagging-strand genes.
Significance Replication and transcription can occur concurrently and use the same DNA template. This can cause genomic instability such as common fragile site instability in eukaryotes, and accelerated evolution of lagging-strand genes in bacteria. Here, we report an underlying mechanism that increases mutation rates of lagging-strand genes in Bacillus subtilis. We find that this process is mediated through the transcription-dependent activity of the Y-family polymerase, PolY1, in transcription-coupled nucleotide excision repair. We find that PolY1, likely through this mechanism, reduces the potentially problematic impacts of specifically lagging-strand transcription on replisome progression and genomic instability. This work shows that gene orientation, together with transcription, can cause differential activities of repair mechanisms, specifically increasing mutagenesis in lagging-strand genes.
The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to ...anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.
The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed ...of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.
The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that serves as a barrier to the environment. During infection, Gram-negative bacteria remodel their OM to promote ...survival and replication within host tissues. Salmonella rely on the PhoPQ two-component regulators to coordinate OM remodeling in response to environmental cues. In a screen for mediators of PhoPQ-regulated OM remodeling in Salmonella Typhimurium, we identified PbgA, a periplasmic domain-containing transmembrane protein, which binds cardiolipin glycerophospholipids near the inner membrane and promotes their PhoPQ-regulated trafficking to the OM. Purified-PbgA oligomers are tetrameric, and the periplasmic domain contains a globular region that binds to the OM in a PhoPQ-dependent manner. Thus, PbgA forms a complex that may bridge the envelope for regulated cardiolipin delivery. PbgA globular region-deleted mutant bacteria are severely attenuated for pathogenesis, suggesting that increased cardiolipin trafficking to the OM is necessary for Salmonella to survive within host tissues that activate PhoPQ.
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•pbgA is a mediator of Salmonella PhoPQ-regulated outer-membrane (OM) remodeling•PhoPQ promotes PbgA-OM interactions and PbgA mediates increases in the OM barrier•The PbgA-globular region is required for regulated increases in OM cardiolipins•The cardiolipin-binding arginines of PbgA are required for cell growth and barrier function
Gram-negative bacteria produce an outer membrane (OM) that serves as a barrier to antibiotics and the immune system. Dalebroux et al. define a conserved transmembrane protein that binds and traffics acidic cardiolipin glycerophospholipids to the bacterial OM to promote the barrier necessary for intracellular survival of Salmonella within host macrophages.
Intracellular Salmonella use a type III secretion system (TTSS) to translocate effector proteins across the phagosome membrane and thus promote vacuole membrane tubulation, resulting in intracellular ...survival. This work demonstrates that the effector SseJ binds the eukaryotic lipid transporter oxysterol binding protein 1 (OSBP1). SseJ directs OSBP1 to the endosomal compartment in a manner dependent on the TTSS located on Salmonella pathogenicity island 2 (SPI2). OSBP1 localization is mediated by both SseJ and another OSBP1-binding SPI2 translocated effector, the deubiquitinase SseL. Deletion of both SseJ and SseL reduced vacuolar integrity with increased bacteria released into the eukaryotic cytoplasm of epithelial cells, indicating that their combined activities are necessary for vacuole membrane stability. Cells knocked down for OSBP1 or deleted for the OSBP1-binding proteins VAPA/B also demonstrate loss of vacuole integrity, consistent with the hypothesis that OSBP1 recruitment is required for SPI2-mediated alterations that promote vacuolar integrity of salmonellae.
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•Salmonella-secreted effector SseJ binds eukaryotic oxysterol binding protein 1•SseJ directs OSBP1 to the endosomal compartment•OSBP1 localization is mediated by both SseJ and another effector, SseL•Deletion of both SseJ and SseL increases bacterial release into the cytoplasm
Kolodziejek et al. demonstrate that the Salmonella-secreted effector SseJ binds eukaryotic oxysterol binding protein 1 (OSBP1). SseJ directs OSBP1 to the endosome in concert with another effector, SseL. Deletion of both SseJ and SseL or OSBP1 knockdown increased bacterial cytoplasmic release, indicating that they are necessary for vacuolar stability.
Fecal microbiota transplantation (FMT) is an investigational treatment for diseases thought to involve alterations in the intestinal microbiota including ulcerative colitis (UC). Case reports have ...described therapeutic benefit of FMT in patients with UC, possibly due to changes in the microbiota. We measured the degree to which the transplanted microbiota engraft following FMT in patients with UC using a donor similarity index (DSI).
Seven patients with mild to moderate UC (UC disease activity index scores 3-10) received a single colonoscopic administration of FMT. Metagenomic sequence data from stool were analyzed using an alignment-free comparison tool, to measure the DSI, and a phylogenetic analysis tool, to characterize taxonomic changes. Clinical, endoscopic, histologic, and fecal calprotectin outcome measures were also collected.
One of 5 patients from whom sequencing data were available achieved the primary endpoint of 50% donor similarity at week 4; an additional 2 patients achieved 40% donor similarity. One patient with 40% donor similarity achieved clinical and histologic remission 1 month after FMT. However, these were lost by 2-3 months, and loss correlated with a decrease in DSI. The remaining patients did not demonstrate clinical response or remission. Histology scores improved in all but 1 patient. No patients remained in remission at 3 months after FMT.
Following a single colonoscopic fecal transplant, a DSI of 40-50% is achieved in about two-thirds of recipients. This level of engraftment correlated with a temporary clinical improvement in only 1/5 patients. Larger sample sizes could further validate this method for measuring engraftment, and changes in transplant frequency or method might improve microbiota engraftment and efficacy.
ClinicalTrials.gov NCT01742754.
Opportunistic infections are often polymicrobial. Two of the most important bacterial opportunistic pathogens of humans, Pseudomonas aeruginosa and Staphylococcus aureus, frequently are coisolated ...from infections of catheters, endotracheal tubes, skin, eyes, and the respiratory tract, including the airways of people with cystic fibrosis (CF). Here, we show that suppression of S. aureus respiration by a P. aeruginosa exoproduct, 4-hydroxy-2-heptylquinoline-N-oxide (HQNO), protects S. aureus during coculture from killing by commonly used aminoglycoside antibiotics such as tobramycin. Furthermore, prolonged growth of S. aureus with either P. aeruginosa or with physiological concentrations of pure HQNO selects for typical S. aureus small-colony variants (SCVs), well known for stable aminoglycoside resistance and persistence in chronic infections, including those found in CF. We detected HQNO in the sputum of CF patients infected with P. aeruginosa, but not in uninfected patients, suggesting that this HQNO-mediated interspecies interaction occurs in CF airways. Thus, in all coinfections with P. aeruginosa, S. aureus may be underappreciated as a pathogen because of the formation of antibiotic-resistant and difficult to detect small-colony variants. Interspecies microbial interactions, analogous to those mediated by HQNO, commonly may alter not only the course of disease and the response to therapy, but also the population structure of bacterial communities that promote the health of host animals, plants, and ecosystems.
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