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The escalating antibiotic resistance crisis poses a major global health threat. Bacteriophage therapy offers a promising alternative for combating multidrug-resistant infections. However, bacterial resistance to phages remains a significant hurdle. Innovative strategies are needed to overcome this challenge. In this study, we developed a phage cocktail based on our phage library, consisting of three phages that suppressed phage resistance of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp). This cocktail capitalized on dual instances of collateral sensitivity, thereby constraining the evolution of phage resistance. The first-layered collateral sensitivity arose from overlapping coverage between capsular polysaccharide (CPS) and lipopolysaccharide (LPS), rendering the bacteria resistant to CPS-binding phages but more susceptible to LPS-binding phages. The second-layered collateral sensitivity resulted from an O serotype switch (from O1 to O2), causing resistance to O1 antigen-binding phages but increasing susceptibility to phages that target the O2 antigen. This dual-layered collateral sensitivity phage cocktail effectively mitigated infection caused by CR-hvKp in mice. Our research highlights the importance of the collateral sensitivity mechanism in counteracting the evolution of phage resistance and offers a sophisticated strategy for configuring phage cocktails to eliminate bacterial resistance.

Eukaryotes are hypothesized to be archaeal-bacterial chimeras. Given the different chemical structures of membrane phospholipids in archaea and bacteria, transformations of membranes during eukaryogenesis that led to the bacterial-type membranes of eukaryotic cells remain a major conundrum. One of the possible intermediates of eukaryogenesis could involve an archaeal-bacterial hybrid membrane. So far, organisms with hybrid membranes have not been discovered, and experimentation on such membranes has been limited. To generate mixed membranes, we reconstructed the archaeal membrane lipid biosynthesis pathway in Escherichia coli, creating three strains that individually produced archaeal lipids ranging from simple, such as DGGGOH (digeranylgeranylglycerol) and archaeol, to complex, such as GDGT (glycerol dialkyl glycerol tetraether). The physiological responses became more pronounced as the hybrid membrane incorporated more complex archaeal membrane lipids. In particular, biosynthesis of GDGT induced a pronounced SOS response, accompanied by cellular filamentation, explosive cell lysis, and ATP accumulation. Thus, bacteria seem to be able to incorporate simple archaeal membrane lipids, such as DGGGOH and archaeol, without major fitness costs, compatible with the involvement of hybrid membranes at the early stages of cell evolution and in eukaryogenesis. By contrast, the acquisition of more complex, structurally diverse membrane lipids, such as GDGT, appears to be strongly deleterious to bacteria, suggesting that this type of lipid is an archaeal innovation.

Evolutionary training of phages can help to counter bacterial resistance evolution. Here, we address whether metapopulation processes can enhance the evolution of phage infectivity. Our experiment with a model bacterium-phage system supported a prediction of long-term fluctuating selection dynamics. Specifically, metapopulations of several small habitats showed greater total infectivity ranges by supporting more diverse phages, compared with single large populations. Crucially, the advantage of several small habitats was conditioned on blocking bacterial dispersal within metapopulations. We conclude that well-designed metapopulation training programs can be useful for quick and easy preparation of cocktail phage materials.

This study identifies avian tuberculosis as a potential cause of mass mortality in wild migratory birds in Inner Mongolia, China. Combining meta-transcriptomic sequencing and histopathological analysis, it reveals one of the rare instances of tuberculosis-associated outbreaks in avian populations. These findings underscore the importance of surveillance on wildlife diseases to mitigate the risk of interspecies transmission of the disease associated pathogens and their broader implications for biodiversity and public health.

The global dissemination of H5 avian influenza viruses represents a significant threat to both human and animal health. In this study, we conducted a genome-wide siRNA library screening against the highly pathogenic H5N1 influenza virus, leading us to the identification of 457 cellular cofactors (441 proviral factors and 16 antiviral factors) involved in the virus replication cycle. Gene Ontology term enrichment analysis revealed that the candidate gene data sets were enriched in gene categories associated with mRNA splicing via spliceosome in the biological process, integral component of membrane in the cellular component, and protein binding in the molecular function. Reactome pathway analysis showed that the immune system (up to 63 genes) was the highest enriched pathway. Subsequent comparisons with four previous siRNA library screenings revealed that the overlapping rates of the involved pathways were 8.53%-62.61%, which were significantly higher than those of the common genes (1.85%-6.24%). Together, our genome-wide siRNA library screening unveiled a panorama of host cellular networks engaged in the regulation of highly pathogenic H5N1 influenza virus replication, which may provide potential targets and strategies for developing novel antiviral countermeasures.

In Sulfolobales cells, transcription of the Ups (UV-inducible pili of Sulfolobus) and Ced (Crenarchaeal system for exchange of DNA) genes is highly induced by DNA damage, and the two systems play key roles in pili-mediated cell aggregation and chromosomal DNA import, respectively. Ups is composed of UpsA, UpsB, UpsE, and UpsF, while Ced is composed of CedA, CedA1, CedA2, and CedB. So far, how DNA is transported by these systems is far from clear. Here, we report three novel components of the Ced and Ups systems in Saccharolobus islandicus REY15A, CedD (SiRe_1715) and CedE (SiRe_2100), paralogs of CedB and CedA, and UpsC (SiRe_1957), a paralog of UpsA/UpsB. We developed a DNA import and export assay method, by which we revealed that CedD, CedE, and UpsC are essential for DNA import, while CedE and UpsC are also involved in DNA export together with CedA1 and Ups. Microscopic analysis revealed that upsC is involved in cell aggregation like other Ups genes. In addition, we found that cedB and cedD co-occur in the Crenarchaeal genomes that lack virB4, an essential component of type IV secretion system. Interestingly, CedB and CedD share homology to different parts of VirB4 N-terminal domain and form stable homo-oligomers in vitro. Collectively, our results indicate that CedD, CedE, and UpsC are integral components of the Ced and Ups systems in Sulfolobales.

The yeast Saccharomyces cerevisiae is a well-studied unicellular eukaryote with a significant role in the biomanufacturing of natural products, biofuels, and bulk and value-added chemicals, as well as the principal model eukaryotic organism utilized for fundamental research. Robust tools for building and optimizing yeast chassis cells were made possible by the quick development of synthetic biology, especially in engineering evolution. In this review, we focused on methods and tools from synthetic biology that are used to design and engineer S. cerevisiae's evolution. A detailed discussion was held regarding transcriptional regulation, template-dependent and template-free approaches. Furthermore, the applications of evolved S. cerevisiae were comprehensively summarized. These included improving environmental stress tolerance and raising cell metabolic performance in the production of biofuels and bulk and value-added chemicals. Finally, the future considerations were briefly discussed.

In Pseudomonas aeruginosa, the dynamic activity of type IV pilus (TFP) is essential for various bacterial behaviors. While PilU is considered a homolog of the TFP disassembling motor PilT, its specific roles remain unclear. Using pilus visualization and single-cell tracking techniques, we characterized TFP dynamics and surface behaviors in wild-type and ΔpilU mutants. We found that ΔpilU cells displayed increased TFP numbers but reduced cell movement and delayed microcolony formation. Interestingly, beyond affecting the twitching motility, ΔpilU cells formed a thick multilayered colony edge on semi-solid surfaces, slowing colony expansion. Cell-cell collision responses changed from touch-turn dominance in wild type to touch-upright dominance in ΔpilU, affecting colony morphology and expansion. These findings expand our understanding of PilU's physiological roles and provide potential targets for developing strategies to control P. aeruginosa biofilm formation and virulence.

Anaerobic methanotrophic (ANME) microbes play a crucial role in the bioprocess of anaerobic oxidation of methane (AOM). However, due to their unculturable status, their diversity is poorly understood. In this study, we established a microfluidics-based epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) to fuse the 16S rRNA gene and mcrA gene to reveal the diversity of ANME microbes (mcrA gene hosts) in three sampling push-cores from the marine cold seep. A total of 3725 16S amplicon sequence variants (ASVs) of the mcrA gene hosts were detected, and classified into 78 genera across 23 phyla. Across all samples, the dominant phyla with high relative abundance (>10%) were the well-known Euryarchaeota, and some bacterial phyla such as Campylobacterota, Proteobacteria, and Chloroflexi; however, the specificity of these associations was not verified. In addition, the compositions of the mcrA gene hosts were significantly different in different layers, where the archaeal hosts increased with the depths of sediments, indicating the carriers of AOM were divergent in depth. Furthermore, the consensus phylogenetic trees of the mcrA gene and the 16S rRNA gene showed congruence in archaea not in bacteria, suggesting the horizontal transfer of the mcrA gene may occur among host members. Finally, some bacterial metagenomes were found to contain the mcrA gene as well as other genes that encode enzymes in the AOM pathway, which prospectively propose the existence of ANME bacteria. This study describes improvements for a potential method for studying the diversity of uncultured functional microbes and broadens our understanding of the diversity of ANMEs.

RNAi technologies have been exploited to control viruses, pests, oomycetes, and fungal phytopathogens that cause disasters in host plants, including many agronomically significant crops. Double-stranded RNA (dsRNA) or small interfering RNA (siRNA) has been applied as a trigger for trans-kingdom RNAi between hosts and fungi. However, it is unclear what process mediates RNA uptake by fungi. In this study, by using live-cell imaging, we determined that exogenously synthesized RNA or small RNA (sRNA) was indiscriminately absorbed into Verticillium dahliae, a notorious pathogenic fungus. Moreover, the application of endocytic inhibitors or deletion of endocytic-related genes reduced RNA uptake efficiency, showing that RNA absorption by fungal cells occurs mainly through endocytosis. In addition, we found that the endocytosed fluorescence-labeled RNAs were partly colocalized with endosome marker genes. Overall, our research concluded that exogenous RNA could be assimilated by V. dahliae through the endocytic pathway. Unraveling this cytological mechanism underlying trans-kingdom RNAi holds significant importance, especially considering the fact that RNAi-based strategies targeting pathogenic fungi are increasingly prevalent in the realm of crop protection.