microLife最新文献

Haloferax volcanii harbours four putative proviruses: Halfvol1, Halfvol2, Halfvol3, and Halfvol4. In this study, we successfully deleted all four provirus genomes, demonstrating, that they are not essential. Transcriptome comparison between this strain (∆Halfvol1-4) and a wild-type strain reveals an increase in archaella and chemotaxis gene expression, resulting in higher swarming motility in ∆Halfvol1-4. Furthermore, ∆Halfvol1-4 cells show an elongated cell shape and a higher resistance to H₂O₂ stress compared to the wild type. RNA-seq also revealed downregulation of CRISPR arrays in the provirus-free strain. Circularised genomes of Halfvol1, Halfvol2, and Halfvol3 were found in the culture supernatant of the wild-type strain. This confirms excision of the proviruses from the chromosome, which seems to happen more efficiently at low temperature (30°C). Electron microscopy revealed potential viral particles in the supernatant, and mass spectrometry analysis confirmed the presence of structural viral proteins of Halfvol1 and Halfvol3 in the isolated virus sample. These observations suggest that these proviruses are active and cause a chronic infection in H. volcanii.

CRISPR-Cas adaptive immune systems in bacteria and archaea enable precise targeting and elimination of invading genetic elements. An inherent feature of these systems is the 'extraneous' CRISPR RNA (ecrRNA), which is produced via the extra repeat in a CRISPR array lacking a corresponding spacer. As ecrRNAs would interact with the Cas machinery yet not direct acquired immunity, they pose a potential barrier to defence. Type II-A CRISPR-Cas systems resolve this barrier through the leader sequence upstream of a CRISPR array, which forms a hairpin structure with the extra repeat that inhibits ecrRNA production. However, the fate of ecrRNAs in other CRISPR types and subtypes remains to be explored. Here, we report that II-C systems likely employ disparate strategies to resolve the ecrRNA due to their distinct configuration in comparison to II-A. Applying bioinformatics analyses to over 650 II-C systems followed by experimental validation, we identified three strategies applicable to these systems: formation of an upstream Rho-independent terminator, formation of a hairpin that sequesters the ecrRNA guide, and mutations in the repeat expected to disrupt ecrRNA formation. These findings expand the list of mechanisms in CRISPR-Cas systems that could resolve the ecrRNA to optimize immune response.

The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans provides defense against mobile genetic elements in the absence of target DNA degradation. In recent studies, Escherichia coli BL21-AI cells with Type IV-A1 CRISPR-Cas activity displayed a heterogeneous colony growth phenotype. Here, we developed a convenient smartphone-mediated automatic remote-controlled time-lapse imaging system (SMARTIS), that enables monitoring of growing bacteria over time. The system's design includes a custom-built imaging box equipped with LED lights, an adjustable heating system and a smartphone that can be remotely controlled using freely available, user-friendly applications. SMARTIS allowed long-term observation of growing colonies and was utilized to analyze different growth behaviors of E. coli cells expressing Type IV-A1 CRISPR ribonucleoproteins. Our findings reveal that heterogeneity in colonies can emerge within hours of initial growth. We further examined the influence of different expression systems on bacterial growth and CRISPR interference activity and demonstrated that the observed heterogeneity of colony-forming units is strongly influenced by plasmid design and backbone identity. This study highlights the importance of careful assessment of heterogenous colony growth dynamics and describes a real-time imaging system with wide applications beyond the study of CRISPR-Cas activity in bacterial hosts.

[This corrects the article DOI: 10.1093/femsml/uqad012.].

Multiple dangerous pathogens from the World Health Organization's priority list possess a plethora of virulence components, including the ability to survive inside macrophages. Often, the pathogens rely on a multi-layered defence strategy in order to defend themselves against the immune system. Here, a minimal model is proposed to study such a strategy. By way of example, we consider the interaction between Pseudomonas aeruginosa and the human host, in which the host and the pathogen counter each other in a back-and-forth interaction. In particular, the pathogen attacks the host, macrophages of the host engulf the pathogen and reduce its access to glucose, the pathogen activates the glyoxylate shunt, which is started by the enzyme isocitrate lyase (Icl), the host inhibits it by itaconic acid, and the pathogen metabolizes itaconic acid using the enzyme succinyl-CoA:itaconate CoA transferase (Ict). The flux through the glyoxylate shunt allows the pathogen to avoid carbon loss and oxidative stress. These functions are of utmost importance inside a phagolysosome. Therefore, the pathogen needs to allocate its limited protein resource between the enzymes Icl and Ict in order to maximize the time integral of a flux through the enzyme Icl. We use both random search and dynamic optimization to identify the enzyme Ict as a cost-effective means of counter-counter-counter-defence and as a possible drug target during the early phase of infection.

Respiratory syncytial virus (RSV), a negative-sense single-stranded RNA virus of the Pneumoviridae family, represents the most important pathogen of lower respiratory tract infections in young infants causing yearly epidemics. RSV is also an important respiratory viral pathogen for older subjects, which is second only to seasonal influenza virus infections. RSV represents a substantial public health burden with respect to morbidity and mortality, particularly in developing countries. Prevention and treatment options would therefore lessen the global disease burden. A formalin-inactivated RSV vaccine in the 1960s induced an enhanced disease upon exposure to natural RSV. After this tragical vaccine failure, it took nearly five decades of intensive research before prevention tools were approved by health authorities. The lead was taken by passive immunity approaches with injected monoclonal antibodies directed against the fusion protein F of RSV. The elucidation of the three-dimensional structure of the F protein revealed pre- and postfusion conformations. Subsequently, structure-based antigen engineering of the F protein paved the way for development of a prophylactic vaccine. In 2023, RSV vaccines were approved for maternal vaccination to protect young infants by placental transfer of antibodies and for vaccination in older subjects. Antiviral drugs that target the RSV fusion process, the RSV replicase, or the cytoplasmic viral factories are in development. Important research papers leading to these developments are reviewed here.

The French Phage Network organizes a scientific meeting every year in which the community of researchers from academia and industry, as well as clinicians participate due to the growing interest in phage therapy. Although centered on giving exposure to future generations of scientists from the French community with senior investigators invited as main speakers, the meeting has also welcomed participants from other countries. Covering almost every aspect of bacteriophage biology, the meeting is an opportunity not only to expose the youngest to a broad range of topics, but also to share their most recent "work in progress" without undergoing a stringent selection process to obtain an oral presentation slot. This report reflects the dynamism of the research field on bacteriophages across multiple disciplines, including molecular and structural biology, ecology, evolution, therapy, and biotechnology.

[This corrects the article DOI: 10.1093/femsml/uqae019.].

Oil reservoirs are society's primary source of hydrocarbons. While microbial communities in industrially exploited oil reservoirs have been investigated in the past, pristine microbial communities in untapped oil reservoirs are little explored, as are distribution patterns of respective genetic signatures. Here, we show that a pristine oil sample contains a complex community consisting of bacteria and fungi for the degradation of hydrocarbons. We identified microorganisms and their pathways for the degradation of methane, n-alkanes, mono-aromatic, and polycyclic aromatic compounds in a metagenome retrieved from biodegraded petroleum encountered in a subsurface reservoir in the Barents Sea. Capitalizing on marker genes from metagenomes and public data mining, we compared the prokaryotes, putative viruses, and putative plasmids of the sampled site to those from 10 other hydrocarbon-associated sites, revealing a shared network of species and genetic elements across the globe. To test for the potential dispersal of the microbes and predicted elements via seawater, we compared our findings to the Tara Ocean dataset, resulting in a broad distribution of prokaryotic and viral signatures. Although frequently shared between hydrocarbon-associated sites, putative plasmids, however, showed little coverage in the Tara Oceans dataset, suggesting an undiscovered mode of transfer between hydrocarbon-affected ecosystems. Based on our analyses, genetic information is globally shared between oil reservoirs and hydrocarbon-associated sites, and we propose that currents and other physical occurrences within the ocean along with deep aquifers are major distributors of prokaryotes and viruses into these subsurface ecosystems.

After exposure to ultraviolet (UV) light, Sulfolobus acidocaldarius cells aggregate in a species-specific manner to exchange DNA and repair double-strand breaks via homologous recombination. The formation of cell-cell interactions is mediated by Ups pili. DNA exchange subsequently occurs through the Crenarchaeal system for exchange of DNA (Ced), which imports DNA. To identify novel players in these processes, we investigated that several genes upregulated after UV exposure, by creating in-frame deletion mutants and performing cell aggregation and DNA exchange assays. This led to the identification of two novel components involved in the Ups and Ced systems: UpsC, a minor pilin of the Ups pili, and CedD, a VirD4-like ATPase essential for DNA import. Altogether, these findings provide new insights into the DNA damage response mechanisms in Sulfolobales.