microLife Pub Date : 2025-07-08 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf015

Anna-Lena Sailer, Julia Wörtz, Victoria Smith, Aris-Edda Stachler, Fabienne Blau, Michelle Daratha, Lisa-Katharina Maier, Thorsten Allers, Anita Marchfelder

{"title":"CRISPR-Cas induced self-targeting identifies key players in archaeal microhomology-mediated end joining.","authors":"Anna-Lena Sailer, Julia Wörtz, Victoria Smith, Aris-Edda Stachler, Fabienne Blau, Michelle Daratha, Lisa-Katharina Maier, Thorsten Allers, Anita Marchfelder","doi":"10.1093/femsml/uqaf015","DOIUrl":"10.1093/femsml/uqaf015","url":null,"abstract":"DNA repair processes are the foundation for genome integrity and survival, especially in extreme environments where DNA damage occurs more frequently and where archaea are found. Nevertheless, first-hand experimental information on repair pathways in archaea is scarce, and assignment of repair proteins is currently largely based on homology. We showed previously that DNA lesions induced by clustered regularly interspaced short palindromic repeats Cas (CRISPR-Cas) self-targeting are repaired by microhomology-mediated end joining (MMEJ). To identify proteins involved in the archaeal MMEJ pathway, we used deletion strains devoid of proteins assigned to the key steps of MMEJ, to examine changes in the repair outcome. In addition, we used aphidicolin to inhibit the activity of the essential PolB1 protein. For the first time, we were thereby able to experimentally identify proteins involved in this repair pathway in the euryarchaeal model organism Haloferax volcanii. This study confirms that Mre11, Rad50, Fen1, PolB1, LigA, and LigN take part in MMEJ, as previously inferred. In addition, we show that Cas1 and Hel308a are also involved in the MMEJ pathway.","PeriodicalId":74189,"journal":{"name":"microLife","volume":"6 ","pages":"uqaf015"},"PeriodicalIF":0.0,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12281630/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144692647","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

"French Phage Network" annual conference: ninth meeting report. “法国噬菌体网络”年会：第九届会议报告。

microLife Pub Date : 2025-07-03 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf014

Fernando Clavijo-Coppens, Deborah M Crepin, Héloïse Croizet, Arthur Planche, Lucile Plumet, Judith Sar, Kandas Traore

{"title":"\"French Phage Network\" annual conference: ninth meeting report.","authors":"Fernando Clavijo-Coppens, Deborah M Crepin, Héloïse Croizet, Arthur Planche, Lucile Plumet, Judith Sar, Kandas Traore","doi":"10.1093/femsml/uqaf014","DOIUrl":"10.1093/femsml/uqaf014","url":null,"abstract":"This meeting report summarizes the scientific activities of the ninth annual conference of Phages.fr, organized by the French Phages network. This year, the conference took place from 12 to 14 November 2024, in Sète, in the south of France. The conference hosted 136 participants from both the public and private sectors, representing 63 French groups and 16 international ones from Austria, Belgium, Finland, Germany, Guinea, Sweden, the UK, and the USA. The meeting brought together both young and senior scientists, offering them the opportunity to share their findings and ideas across four main topics: Ecology and Evolution, Phage-Host Interaction, Structure and Assembly, and Applications in Therapy and Biotechnology. For the first time, Phages.fr also offered a special session dedicated to the social and human sciences applied to microbiology. Over the 3 days, a total of 62 presentations were given (20 oral presentations and 42 posters), and five invited speakers delivered exceptional lectures introducing each session. The ninth annual symposium concluded with a public lecture titled \"Viruses of Bacteria: New Allies in Human and Agricultural Health.\" The lecture aimed to raise public awareness about the therapeutic potential of phages in combating harmful bacteria that affect human and plant health, as well as their role in food safety.","PeriodicalId":74189,"journal":{"name":"microLife","volume":"6 ","pages":"uqaf014"},"PeriodicalIF":0.0,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12254948/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144627966","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Microbes are life, the biological core of One Health and Health in All Policies. 微生物是生命，是“一个健康”和“健康在所有政策”的生物学核心。

microLife Pub Date : 2025-06-28 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf012

Kenneth Timmis, Fernando Baquero, James K Timmis, Margaret Douglas

引用次数: 0

CRISPR-Cas in actinomycetes: still a lot to be discovered. 放线菌中的CRISPR-Cas：仍有很多有待发现。

microLife Pub Date : 2025-06-12 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf010

Lena Mitousis, Ewa Musiol-Kroll, Wolfgang Wohlleben

{"title":"CRISPR-Cas in actinomycetes: still a lot to be discovered.","authors":"Lena Mitousis, Ewa Musiol-Kroll, Wolfgang Wohlleben","doi":"10.1093/femsml/uqaf010","DOIUrl":"10.1093/femsml/uqaf010","url":null,"abstract":"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.","PeriodicalId":74189,"journal":{"name":"microLife","volume":"6 ","pages":"uqaf010"},"PeriodicalIF":0.0,"publicationDate":"2025-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12231136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Heterodimerization of staphylococcal phage φ2638A endolysin isoforms and their functional role in bacterial lysis. 葡萄球菌噬菌体φ2638A内溶素异构体的异源二聚化及其在细菌裂解中的功能作用。

microLife Pub Date : 2025-06-10 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf011

Léa V Zinsli, Anna M Sobieraj, Jiemin Du, Patrick Ernst, Susanne Meile, Samuel Kilcher, Cedric Iseli, Anja P Keller, Birgit Dreier, Peer R E Mittl, Andreas Plückthun, Martin J Loessner, Mathias Schmelcher, Matthew Dunne

{"title":"Heterodimerization of staphylococcal phage φ2638A endolysin isoforms and their functional role in bacterial lysis.","authors":"Léa V Zinsli, Anna M Sobieraj, Jiemin Du, Patrick Ernst, Susanne Meile, Samuel Kilcher, Cedric Iseli, Anja P Keller, Birgit Dreier, Peer R E Mittl, Andreas Plückthun, Martin J Loessner, Mathias Schmelcher, Matthew Dunne","doi":"10.1093/femsml/uqaf011","DOIUrl":"10.1093/femsml/uqaf011","url":null,"abstract":"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.","PeriodicalId":74189,"journal":{"name":"microLife","volume":"6 ","pages":"uqaf011"},"PeriodicalIF":0.0,"publicationDate":"2025-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12203907/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144531454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Metabolic pathway analysis of the methylcitrate cycle in bacteria and fungi identifies methylcitrate synthase as an antiinfective drug target. 甲基柠檬酸循环在细菌和真菌中的代谢途径分析确定了甲基柠檬酸合成酶作为抗感染药物的靶点。

microLife Pub Date : 2025-05-19 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf009

Lukas Korn, Matthias Brock, Stefan Schuster

{"title":"Metabolic pathway analysis of the methylcitrate cycle in bacteria and fungi identifies methylcitrate synthase as an antiinfective drug target.","authors":"Lukas Korn, Matthias Brock, Stefan Schuster","doi":"10.1093/femsml/uqaf009","DOIUrl":"10.1093/femsml/uqaf009","url":null,"abstract":"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.","PeriodicalId":74189,"journal":{"name":"microLife","volume":"6 ","pages":"uqaf009"},"PeriodicalIF":0.0,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12125574/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144201015","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Provirus deletion from Haloferax volcanii affects motility, stress resistance, and CRISPR RNA expression. 火山盐黄貂的原病毒缺失影响运动性、抗逆性和CRISPR RNA表达。

microLife Pub Date : 2025-05-19 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf008

Nadia Di Cianni, Simon Bolsinger, Jutta Brendel, Monika Raabe, Sabine König, Laura Mitchell, Thorsten Bischler, Tom Gräfenhan, Clarissa Read, Susanne Erdmann, Thorsten Allers, Paul Walther, Henning Urlaub, Mike Dyall-Smith, Friedhelm Pfeiffer, Anita Marchfelder

{"title":"Provirus deletion from Haloferax volcanii affects motility, stress resistance, and CRISPR RNA expression.","authors":"Nadia Di Cianni, Simon Bolsinger, Jutta Brendel, Monika Raabe, Sabine König, Laura Mitchell, Thorsten Bischler, Tom Gräfenhan, Clarissa Read, Susanne Erdmann, Thorsten Allers, Paul Walther, Henning Urlaub, Mike Dyall-Smith, Friedhelm Pfeiffer, Anita Marchfelder","doi":"10.1093/femsml/uqaf008","DOIUrl":"10.1093/femsml/uqaf008","url":null,"abstract":"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.","PeriodicalId":74189,"journal":{"name":"microLife","volume":"6 ","pages":"uqaf008"},"PeriodicalIF":0.0,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12091111/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144112610","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Disparate mechanisms counteract extraneous CRISPR RNA production in type II-C CRISPR-Cas systems. 在II-C型CRISPR- cas系统中，不同的机制抵消了外来CRISPR RNA的产生。

microLife Pub Date : 2025-05-14 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf007

Maximilian Feussner, Angela Migur, Alexander Mitrofanov, Omer S Alkhnbashi, Rolf Backofen, Chase L Beisel, Zasha Weinberg

{"title":"Disparate mechanisms counteract extraneous CRISPR RNA production in type II-C CRISPR-Cas systems.","authors":"Maximilian Feussner, Angela Migur, Alexander Mitrofanov, Omer S Alkhnbashi, Rolf Backofen, Chase L Beisel, Zasha Weinberg","doi":"10.1093/femsml/uqaf007","DOIUrl":"https://doi.org/10.1093/femsml/uqaf007","url":null,"abstract":"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.","PeriodicalId":74189,"journal":{"name":"microLife","volume":"6 ","pages":"uqaf007"},"PeriodicalIF":0.0,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12080349/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144082598","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Real-time imaging of bacterial colony growth dynamics for cells with Type IV-A1 CRISPR-Cas activity. 具有IV-A1型CRISPR-Cas活性细胞的细菌集落生长动态的实时成像。

microLife Pub Date : 2025-04-01 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf006

Selina Rust, Lennart Randau

{"title":"Real-time imaging of bacterial colony growth dynamics for cells with Type IV-A1 CRISPR-Cas activity.","authors":"Selina Rust, Lennart Randau","doi":"10.1093/femsml/uqaf006","DOIUrl":"10.1093/femsml/uqaf006","url":null,"abstract":"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.","PeriodicalId":74189,"journal":{"name":"microLife","volume":"6 ","pages":"uqaf006"},"PeriodicalIF":0.0,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11995694/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144030993","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Correction to: Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics. 更正：通过核糖体分析和蛋白质基因组学揭示植物共生体Sinorhizobium meliloti的小蛋白质组

microLife Pub Date : 2025-03-28 eCollection Date: 2025-01-01 DOI: 10.1093/femsml/uqaf004

引用次数: 0

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