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1. Bacteriophage T4 Escapes CRISPR Attack by Minihomology Recombination and Repair

   https://doi.org/10.1128/mBio.01361-21  

   Wu, Xiaorong ; Zhu, Jingen ; Tao, Pan ; Rao, Venigalla B. ( June 2021 , mBio)

   Hatfull, Graham F. (Ed.)

   ABSTRACT Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) is a bacterial defense system that inactivates the invading phage genome by introducing double-strand breaks at targeted sequences. While the mechanisms of CRISPR defense have been extensively investigated, the counterdefense mechanisms employed by phages are poorly understood. Here, we report a novel counterdefense mechanism by which phage T4 restores the genomes broken by CRISPR cleavages. Catalyzed by the phage-encoded recombinase UvsX, this mechanism pairs very short stretches of sequence identity (minihomology sites), as few as 3 or 4 nucleotides in the flanking regions of the cleaved site, allowing replication, repair, and stitching of genomic fragments. Consequently, a series of deletions are created at the targeted site, making the progeny genomes completely resistant to CRISPR attack. Our results demonstrate that this is a general mechanism operating against both type II (Cas9) and type V (Cas12a) CRISPR-Cas systems. These studies uncovered a new type of counterdefense mechanism evolved by T4 phage where subtle functional tuning of preexisting DNA metabolism leads to profound impact on phage survival. IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria and use them as replication factories to assemble progeny phages. Bacteria have evolved powerful defense mechanisms to destroy the invading phages by severing their genomes soon after entry into cells. We discovered a counterdefense mechanism evolved by phage T4 to stitch back the broken genomes and restore viral infection. In this process, a small amount of genetic material is deleted or another mutation is introduced, making the phage resistant to future bacterial attack. The mutant virus might also gain survival advantages against other restriction conditions or DNA damaging events. Thus, bacterial attack not only triggers counterdefenses but also provides opportunities to generate more fit phages. Such defense and counterdefense mechanisms over the millennia led to the extraordinary diversity and the greatest abundance of bacteriophages on Earth. Understanding these mechanisms will open new avenues for engineering recombinant phages for biomedical applications. 

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2. Cylindrospermopsis raciborskii Virus and host: genomic characterization and ecological relevance

   https://doi.org/10.1111/1462-2920.14425  

   Martin, Robbie M. ; Moniruzzaman, Mohammad ; Mucci, Nicholas C. ; Willis, Anusuya ; Woodhouse, Jason N. ; Xian, Yuejiao ; Xiao, Chuan ; Brussaard, Corina P. D. ; Wilhelm, Steven W. ( October 2018 , Environmental Microbiology)

   Cylindrospermopsis (Raphidiopsis) raciborskiiis an invasive, filamentous, nitrogen‐fixing cyanobacterium that forms frequent blooms in freshwater habitats. While viruses play key roles in regulating the abundance, production and diversity of their hosts in aquatic ecosystems, the role(s) of viruses in the ecology ofC. raciborskiiis almost unexplored. Progress in this field has been hindered by the absence of a characterized virus–host system inC. raciborskii. To bridge this gap, we sequenced the genome of CrV‐01T, a previously isolated cyanosiphovirus, and its host,C. raciborskiistrain Cr2010. Analyses suggest that CrV‐01T represents a distinct clade of siphoviruses infecting, and perhaps lysogenizing, filamentous cyanobacteria. Its genome contains unique features that include an intact CRISPR array and a 12 kb inverted duplication. Evidence suggests CrV‐01T recently gained the ability to infect Cr2010 and recently lost the ability to form lysogens. The cyanobacterial host contains a CRISPR‐Cas system with CRISPR spacers matching protospacers within the inverted duplication of the CrV‐01T genome. Examination of metagenomes demonstrates that viruses with high genetic identity to CrV‐01T, but lacking the inverted duplication, are present inC. raciborskiiblooms in Australia. The unique genomic features of the CrV/Cr2010 system offers opportunities to investigate in more detail virus–host interactions in an ecologically important bloom‐forming cyanobacterium.

    

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3. Targeted assemblies of cas1 suggest CRISPR-Cas’s response to soil warming

   https://doi.org/10.1038/s41396-020-0635-1  

   Wu, Ruonan ; Chai, Benli ; Cole, James R. ; Gunturu, Santosh K. ; Guo, Xue ; Tian, Renmao ; Gu, Ji-Dong ; Zhou, Jizhong ; Tiedje, James M. ( March 2020 , The ISME Journal)

   There is an increasing interest in the clustered regularly interspaced short palindromic repeats CRISPR-associated protein (CRISPR-Cas) system to reveal potential virus–host dynamics. The universal and most conserved Cas protein, cas1 is an ideal marker to elucidate CRISPR-Cas ecology. We constructed eight Hidden Markov Models (HMMs) and assembled cas1 directly from metagenomes by a targeted-gene assembler, Xander, to improve detection capacity and resolve the diverse CRISPR-Cas systems. The eight HMMs were first validated by recovering all 17 cas1 subtypes from the simulated metagenome generated from 91 prokaryotic genomes across 11 phyla. We challenged the targeted method with 48 metagenomes from a tallgrass prairie in Central Oklahoma recovering 3394 cas1. Among those, 88 were near full length, 5 times more than in de-novo assemblies from the Oklahoma metagenomes. To validate the host assignment by cas1, the targeted-assembled cas1 was mapped to the de-novo assembled contigs. All the phylum assignments of those mapped contigs were assigned independent of CRISPR-Cas genes on the same contigs and consistent with the host taxonomies predicted by the mapped cas1. We then investigated whether 8 years of soil warming altered cas1 prevalence within the communities. A shift in microbial abundances was observed during the year with the biggest temperature differential (mean 4.16 °C above ambient). cas1 prevalence increased and even in the phyla with decreased microbial abundances over the next 3 years, suggesting increasing virus–host interactions in response to soil warming. This targeted method provides an alternative means to effectively mine cas1 from metagenomes and uncover the host communities.

    

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4. Life strategies for Aminicenantia in subseafloor oceanic crust

   https://doi.org/10.1038/s41396-023-01454-5  

   Booker, Anne E. ; D’Angelo, Timothy ; Adams-Beyea, Annabelle ; Brown, Julia M. ; Nigro, Olivia ; Rappé, Michael S. ; Stepanauskas, Ramunas ; Orcutt, Beth N. ( June 2023 , The ISME Journal)

   After decades studying the microbial “deep biosphere” in subseafloor oceanic crust, the growth and life strategies in this anoxic, low energy habitat remain poorly described. Using both single cell genomics and metagenomics, we reveal the life strategies of two distinct lineages of uncultivated Aminicenantia bacteria from the basaltic subseafloor oceanic crust of the eastern flank of the Juan de Fuca Ridge. Both lineages appear adapted to scavenge organic carbon, as each have genetic potential to catabolize amino acids and fatty acids, aligning with previous Aminicenantia reports. Given the organic carbon limitation in this habitat, seawater recharge and necromass may be important carbon sources for heterotrophic microorganisms inhabiting the ocean crust. Both lineages generate ATP via several mechanisms including substrate-level phosphorylation, anaerobic respiration, and electron bifurcation driving an Rnf ion translocation membrane complex. Genomic comparisons suggest these Aminicenantia transfer electrons extracellularly, perhaps to iron or sulfur oxides consistent with mineralogy of this site. One lineage, called JdFR-78, has small genomes that are basal to the Aminicenantia class and potentially use “primordial” siroheme biosynthetic intermediates for heme synthesis, suggesting this lineage retain characteristics of early evolved life. Lineage JdFR-78 contains CRISPR-Cas defenses to evade viruses, while other lineages contain prophage that may help prevent super-infection or no detectable viral defenses. Overall, genomic evidence points to Aminicenantia being well adapted to oceanic crust environments by taking advantage of simple organic molecules and extracellular electron transport.

    

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5. Interactions between ultraviolet radiation exposure and phosphorus limitation in the marine nitrogen‐fixing cyanobacteria Trichodesmium and Crocosphaera

   https://doi.org/10.1002/lno.11304  

   Zhu, Zhu ; Fu, Feixue ; Qu, Pingping ; Mak, Esther Wing Kwan ; Jiang, Haibo ; Zhang, Ruifeng ; Zhu, Zhuoyi ; Gao, Kunshan ; Hutchins, David A. ( August 2019 , Limnology and Oceanography)

   Increased stratification and mixed layer shoaling of the surface ocean resulting from warming can lead to exposure of marine dinitrogen (N₂)‐fixing cyanobacteria to higher levels of inhibitory ultraviolet (UV) radiation. These same processes also reduce vertically advected supplies of the potentially limiting nutrient phosphorus (P) to N₂fixers. It is currently unknown how UV inhibition and P limitation interact to affect the biogeochemical cycles of nitrogen and carbon in these biogeochemically critical microbes. We investigated the responses of the important and widespread marine N₂‐fixing cyanobacteriaCrocosphaera(strain WH0005) andTrichodesmium(strains IMS 101 and GBR) to UV‐A and UV‐B under P‐replete and P‐limited conditions. Growth, N₂fixation, and carbon dioxide (CO₂) fixation rates ofTrichodesmiumIMS 101 andCrocosphaerawere negatively affected by UV exposure. This inhibition was greater forTrichodesmiumIMS 101 than forCrocosphaera, which fixes N₂only during the night and so avoids direct UV damage. Negative effects of UV on both IMS 101 andCrocosphaerawere less in P‐limited cultures than in P‐replete cultures. In contrast, no UV inhibition was observed in GBR, regardless of P availability. UV inhibition was related to different amounts of UV‐absorbing compounds produced by these isolates. Responses to UV radiation and P availability interactions were taxon‐specific, but our results indicated that in general, UV radiation effects onTrichodesmiumandCrocosphaerarange from negative to neutral. UV inhibition and its interactions with P limitation may thus have a substantial influence on the present day and future nitrogen and carbon cycles of the ocean.

    

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