Fallonzhang5696

s at which plant microbiomes are surveyed. In our study, phyllosphere tissues housed increasingly distinct microbial assemblages as plants aged, indicating that plants can be considered collections of tissue habitats in which microbial colonists-natural or synthetic-are established with differing success. Relationships between host genes and community diversity might vary depending on when samples are collected, given that assemblages grew more diverse as plants aged. Both spatial and temporal trends weakened when colonists were grouped by family, suggesting that functional rather than taxonomic profiling will be necessary to understand the basis for differences in colonization success.Viral genome sequencing has guided our understanding of the spread and extent of genetic diversity of SARS-CoV-2 during the COVID-19 pandemic. SARS-CoV-2 viral genomes are usually sequenced from nasopharyngeal swabs of individual patients to track viral spread. Recently, RT-qPCR of municipal wastewater has been used to quantify the abundance of SARS-CoV-2 in several regions globally. However, metatranscriptomic sequencing of wastewater can be used to profile the viral genetic diversity across infected communities. Here, we sequenced RNA directly from sewage collected by municipal utility districts in the San Francisco Bay Area to generate complete and nearly complete SARS-CoV-2 genomes. The major consensus SARS-CoV-2 genotypes detected in the sewage were identical to clinical genomes from the region. Using a pipeline for single nucleotide variant calling in a metagenomic context, we characterized minor SARS-CoV-2 alleles in the wastewater and detected viral genotypes which were also found within clinical genomes throughout California. Observed wastewater variants were more similar to local California patient-derived genotypes than they were to those from other regions within the United States or globally. Additional variants detected in wastewater have only been identified in genomes from patients sampled outside California, indicating that wastewater sequencing can provide evidence for recent introductions of viral lineages before they are detected by local clinical sequencing. These results demonstrate that epidemiological surveillance through wastewater sequencing can aid in tracking exact viral strains in an epidemic context.The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome, which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knockdown. Depletion of DHX38, WDR70, and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, and CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of a subset of cellular mRNAs, including some with selectively retained introns. Thus, CRNKL1-dependent nuclear retention is a novel cellular mechanism for the regulation of cytoplasmic levels of intron-retaining HIV-1 mRNAs, which HIV-1 may have harnessed to direct its complex splicing pattern.IMPORTANCE To regulate its complex splicing pattern, HIV-1 uses the adaptor protein Rev to shuttle unspliced or partially spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev, these RNAs are retained in the nucleus, but it is unclear why. Here we identify cellular proteins whose depletion enhances cytoplasmic levels of the HIV-1 unspliced RNA. Depletion of one of them, CRNKL1, also increases cytoplasmic levels of a subset of intron-retaining cellular mRNA, suggesting that CRNKL1-dependent nuclear retention may be a basic cellular mechanism exploited by HIV-1.Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can enter into a persistent state that confers resistance to antibacterial agents. Many observations suggest that persistent M. tuberculosis cells also evade the antimycobacterial immune mechanisms, thereby reducing the effectiveness of the current tuberculosis vaccine. Understanding the factors that contribute to persistence may enable the rational design of vaccines that stimulate effective immune killing mechanisms against persister cells. Independent mutations targeting the methionine and arginine biosynthetic pathways are bactericidal for M. tuberculosis in mice. However, in this study, we discovered that the addition of leucine and pantothenate auxotrophy altered the bactericidality of methionine auxotrophy. Whereas the leucine/pantothenate/methionine auxotrophic M. tuberculosis strain H37Rv ΔleuCD ΔpanCD ΔmetA was eliminated in immunocompetent mice, this strain persisted in multiple organs of immunodeficient Rag1-/- mice for at leasveness of current tuberculosis vaccines. Understanding the factors that contribute to persistence would enable the rational design of vaccines effective against persisters. We previously generated two attenuated, triple-auxotrophic M. tuberculosis strains that are safe to use in a biosafety level 2 laboratory. Herein, we discovered that the triple-auxotrophic strain H37Rv ΔleuCD ΔpanCD ΔmetA persisted in immunodeficient Rag1-/- mice, which lack adaptive immunity, but not in immunocompetent mice. The conditional persistence of this auxotrophic mutant, which is susceptible to the sterilizing effect of the adaptive immune response over time, provides an important tool to dissect the mycobactericidal effector mechanisms mediated by adaptive immunity. Furthermore, because of its remarkable safety attributes, this auxotrophic mutant can potentially be used to develop a practical human challenge model to facilitate vaccine development.It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes, but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback and then barcoded for tracking via sequencing (i.e., random deletion library sequencing [RanDeL-seq]). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1's cis and trans elements. The resulting landscape recapitulated HIV-1's known cis-acting elements (i.e., long terminal repeat [LTR], Ψ, and Rev response element [RRE]) and, surprisingly, indicatedletion mutants that conditionally replicate. Identifying and engineering DIPs require that viral cis- and trans-acting elements be accurately mapped. Here, we introduce a high-throughput method (random deletion library sequencing [RanDeL-seq]) to comprehensively map cis- and trans-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central polypurine tract (cPPT), and identified a new cis region proximal to the Rev responsive element (RRE). RanDeL-seq also identified regions of Zika virus required for replication and packaging. RanDeL-seq is a versatile and comprehensive technique to rapidly map cis and trans regions of a genome.Growth of plastic waste in the natural environment, and in particular in the oceans, has raised the accumulation of polystyrene and other polymeric species in eukyarotic cells to the level of a credible and systemic threat. Oligomers, the smallest products of polymer degradation or incomplete polymerization reactions, are the first species to leach out of macroscopic or nanoscopic plastic materials. However, the fundamental mechanisms of interaction between oligomers and polymers with the different cell components are yet to be elucidated. Simulations performed on lipid bilayers showed changes in membrane mechanical properties induced by polystyrene, but experimental results performed on cell membranes or on cell membrane models are still missing. We focus here on understanding how embedded styrene oligomers affect the phase behavior of model membranes using a combination of scattering, fluorescence, and calorimetric techniques. Our results show that styrene oligomers disrupt the phase behavior of lipid membranes, modifying the thermodynamics of the transition through a spatial modulation of lipid composition.Glacial landforms, including lobate debris aprons, are a global water ice reservoir on Mars preserving ice from past periods when high orbital obliquity permitted nonpolar ice accumulation. Numerous studies have noted morphological similarities between lobate debris aprons and terrestrial debris-covered glaciers, an interpretation supported by radar observations. On Earth and Mars, these landforms consist of a core of flowing ice covered by a rocky lag. Terrestrial debris-covered glaciers advance in response to climate forcing driven by obliquity-paced changes to ice mass balance. However, on Mars, it is not known whether glacial landforms emplaced over the past 300 to 800 formed during a single, long deposition event or during multiple glaciations. Here, we show that boulders atop 45 lobate debris aprons exhibit no evidence of monotonic comminution but are clustered into bands that become more numerous with increasing latitude, debris apron length, and pole-facing flow orientation. Boulder bands are prominent at glacier headwalls, consistent with debris accumulation during the current Martian interglacial. Dyngo-4a purchase Terrestrial glacier boulder bands occur near flow discontinuities caused by obliquity-driven hiatuses in ice accumulation, forming internal debris layers. By analogy, we suggest that Martian lobate debris aprons experienced multiple cycles of ice deposition, followed by ice destabilization in the accumulation zone, leading to boulder-dominated lenses and subsequent ice deposition and continued flow. Correlation between latitude and boulder clustering suggests that ice mass-balance works across global scales on Mars. Lobate debris aprons may preserve ice spanning multiple glacial/interglacial cycles, extending Mars climate records back hundreds of millions of years.In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates Nδ and Nω' of Nω-methyl-l-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the monoiron cofactor in the enzyme's C-terminal cupin domain, which promotes the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. We leveraged our recent observation that the N-oxygenating µ-peroxodiiron(III/III) intermediate can form in the HO-like domain after the apo protein self-assembles its diiron(II/II) cofactor to solve structures of SznF with both of its iron cofactors bound. These structures of a biochemically validated member of the emerging heme-oxygenase-like diiron oxidase and oxygenase (HDO) superfamily with intact diiron cofactor reveal both the large-scale conformational change required to assemble the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability-a trait shared by the other validated HDOs.