microLife最新文献

Actinomycetes are important producers of valuable natural products that are applied in medicine or industry. The enzymes necessary for the synthesis of those compounds are encoded in biosynthetic gene clusters (BGCs) in the genome. However, the discovery of new natural products or the improvement of production levels can be hindered by difficulties in genetic manipulation, since standard methods often do not or not efficiently work in actinomycetes. One possible explanation for this could be the presence of nucleic acid defense systems such as CRISPR-Cas. Even though there is a lot of research published about CRISPR-Cas systems in general, the knowledge about the function of CRISPR-Cas in actinomycetes is very limited. Based on sequence data it is known that CRISPR-Cas systems occur in around half of all sequenced actinobacterial genomes. Moreover, in silico analyses of those systems have led to the discovery of new subtypes. The few examples of experimental evidence of CRISPR-Cas activity in vivo or in vitro, however, point to some special features, regarding crRNA maturation or life-cycle dependent CRISPR-Cas activity. This short review draws attention to this neglected research area and highlights the available data about CRISPR-Cas in actinomycetes.

Bacteriophage endolysins targeting Gram-positive bacteria typically feature a modular architecture of one or more enzymatically active domains (EADs) and cell wall binding domains (CBDs). Several endolysins also feature internal translational start sites (iTSSs) that produce short variant (SV) isoforms alongside the full-length (FL) endolysin. While the lytic activity of endolysins and their isoforms has been extensively studied as exogenous agents, the purpose behind producing the SV isoform during the phage infection cycle remains to be explored. In this study, we used staphylococcal phage φ2638A as a model to determine the interplay between its FL endolysin, Ply2638A, and its SV isoform during phage infection. X-ray crystallography structures and AlphaFold-generated models enabled elucidation of individual functions of the M23 endopeptidase, central amidase, and SH3b domains of Ply2638A. Production of the SV isoform (amidase and SH3b) was confirmed during phage infection and shown to form a heterodimer complex with Ply2638A via interamidase domain interactions. Using genetically engineered phage variants, we show that production of both isoforms provides an advantage during phage infection as phages producing only one isoform presented delayed progeny phage release as well as impaired lytic activity, which was partly restored through complementation of the missing isoform protein. Interestingly, when applied as an antimicrobial against Staphylococcus aureus in culture, the activity of Ply2638A remained constant regardless of SV isoform complementation. We propose that the SV isoform enhances the efficiency of cell lysis and progeny release at the end of the lytic cycle, providing a functional explanation for iTSSs conservation across diverse phage genomes.

The tricarboxylic acid (TCA) cycle is well known as a crucial pathway in central metabolism in many organisms. A less known analogous pathway is the methylcitrate cycle (MCC). It is present in various fungi such as Aspergillus species and bacteria such as Escherichia coli, with some of them being pathogenic. The MCC catalyzes an alpha-oxidation of propionyl-CoA to pyruvate and is of interest in view of biotechnology and pharmacology. To elucidate the potential interaction of the MCC with other central metabolic pathways, we investigated the MCC by Elementary-flux-mode analysis. We first established a reaction network model, using information from both the KEGG database and literature. This reaction network contains enzymes of the MCC as well as of the TCA cycle, glyoxylate shunt, and carbon source-utilizing pathways, such as amino acid degradation. The network was then used to calculate the elementary flux modes (EFMs) by using the simulation software Metatool 4.3. We identified 76 EFMs, with 39 of them containing the MCC. In this way, some previously known pathways were confirmed theoretically and, additionally, some new EFMs were discovered. Among these, a different, but shorter version of the MCC was identified. The EFMs were systematically analyzed with respect to their ATP yield and the robustness of the network was computed. Predictions on the impact of enzyme deletion or inhibition on the network were made. From these analyses and based on the absence of the MCC in humans, we conclude that the methylcitrate synthase represents a promising drug target against various human pathogens.

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.