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April 21, 2020  |  

Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1

Pear ring rot disease, mainly caused by Botryosphaeria dothidea, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (Botryosphaeria dothidea chrysovirus 1) and BdPV1 (Botryosphaeria dothidea partitivirus 1) influence the biological characteristics of B. dothidea strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between B. dothidea and mycovirus to clarify the pathogenic mechanisms of B. dothidea and hypovirulence of B. dothidea in pear. A high-quality full-length genome sequence of the B. dothidea LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34?Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. B. dothidea was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection. Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in B. dothidea defense, we constructed four small RNA libraries and sequenced B. dothidea micro-like RNAs (Bd-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel Bd-milRNAs were identified, of which 161 conserved and 20 novel Bd-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed Bd-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of B. dothidea strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in B. dothidea through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.


October 23, 2019  |  

SAPTA: a new design tool for improving TALE nuclease activity.

Transcription activator-like effector nucleases (TALENs) have become a powerful tool for genome editing due to the simple code linking the amino acid sequences of their DNA-binding domains to TALEN nucleotide targets. While the initial TALEN-design guidelines are very useful, user-friendly tools defining optimal TALEN designs for robust genome editing need to be developed. Here we evaluated existing guidelines and developed new design guidelines for TALENs based on 205 TALENs tested, and established the scoring algorithm for predicting TALEN activity (SAPTA) as a new online design tool. For any input gene of interest, SAPTA gives a ranked list of potential TALEN target sites, facilitating the selection of optimal TALEN pairs based on predicted activity. SAPTA-based TALEN designs increased the average intracellular TALEN monomer activity by >3-fold, and resulted in an average endogenous gene-modification frequency of 39% for TALENs containing the repeat variable di-residue NK that favors specificity rather than activity. It is expected that SAPTA will become a useful and flexible tool for designing highly active TALENs for genome-editing applications. SAPTA can be accessed via the website at http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html.


October 23, 2019  |  

Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice.

Bacterial blight of rice is caused by the ?-proteobacterium Xanthomonas oryzae pv. oryzae, which utilizes a group of type III TAL (transcription activator-like) effectors to induce host gene expression and condition host susceptibility. Five SWEET genes are functionally redundant to support bacterial disease, but only two were experimentally proven targets of natural TAL effectors. Here, we report the identification of the sucrose transporter gene OsSWEET13 as the disease-susceptibility gene for PthXo2 and the existence of cryptic recessive resistance to PthXo2-dependent X. oryzae pv. oryzae due to promoter variations of OsSWEET13 in japonica rice. PthXo2-containing strains induce OsSWEET13 in indica rice IR24 due to the presence of an unpredicted and undescribed effector binding site not present in the alleles in japonica rice Nipponbare and Kitaake. The specificity of effector-associated gene induction and disease susceptibility is attributable to a single nucleotide polymorphism (SNP), which is also found in a polymorphic allele of OsSWEET13 known as the recessive resistance gene xa25 from the rice cultivar Minghui 63. The mutation of OsSWEET13 with CRISPR/Cas9 technology further corroborates the requirement of OsSWEET13 expression for the state of PthXo2-dependent disease susceptibility to X. oryzae pv. oryzae. Gene profiling of a collection of 104 strains revealed OsSWEET13 induction by 42 isolates of X. oryzae pv. oryzae. Heterologous expression of OsSWEET13 in Nicotiana benthamiana leaf cells elevates sucrose concentrations in the apoplasm. The results corroborate a model whereby X. oryzae pv. oryzae enhances the release of sucrose from host cells in order to exploit the host resources.© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.


October 23, 2019  |  

Identification and expression analysis of chemosensory genes in the citrus fruit fly Bactrocera (Tetradacus) minax

The citrus fruit fly Bactrocera (Tetradacus) minax is a major and devastating agricultural pest in Asian subtropical countries. Previous studies have shown that B. minax interacts with hosts via an efficient chemosensory system. However, knowledge regarding the molecular components of the B. minax chemosensory system has not yet been well established. Herein, based on our newly generated whole-genome dataset for B. minax and by comparison with the characterized genomes of 6 other fruit fly species, we identified, for the first time, a total of 25 putative odorant-binding receptors (OBPs), 4 single-copy chemosensory proteins (CSPs) and 53 candidate odorant receptors (ORs). To further survey the expression of these candidate genes, the transcriptomes from three developmental stages (larvae, pupae and adults) of B. minax and Bactrocera dorsalis were analyzed. We found that 1) at the adult developmental stage, there were 14 highly expressed OBPs (FPKM>100) in B. dorsalis and 7 highly expressed OBPs in B. minax; 2) the expression of CSP3 and CSP4 in adult B. dorsalis was higher than that in B. minax; and 3) most of the OR genes exhibited low expression at the three developmental stages in both species. This study on the identification of the chemosensory system of B. minax not only enriches the existing research on insect olfactory receptors but also provides new targets for preventative control and ecological regulation of B. minax in the future.


October 23, 2019  |  

A knowledge-based molecular screen uncovers a broad-spectrum OsSWEET14 resistance allele to bacterial blight from wild rice.

Transcription activator-like (TAL) effectors are type III-delivered transcription factors that enhance the virulence of plant pathogenic Xanthomonas species through the activation of host susceptibility (S) genes. TAL effectors recognize their DNA target(s) via a partially degenerate code, whereby modular repeats in the TAL effector bind to nucleotide sequences in the host promoter. Although this knowledge has greatly facilitated our power to identify new S genes, it can also be easily used to screen plant genomes for variations in TAL effector target sequences and to predict for loss-of-function gene candidates in silico. In a proof-of-principle experiment, we screened a germplasm of 169 rice accessions for polymorphism in the promoter of the major bacterial blight susceptibility S gene OsSWEET14, which encodes a sugar transporter targeted by numerous strains of Xanthomonas oryzae pv. oryzae. We identified a single allele with a deletion of 18 bp overlapping with the binding sites targeted by several TAL effectors known to activate the gene. We show that this allele, which we call xa41(t), confers resistance against half of the tested Xoo strains, representative of various geographic origins and genetic lineages, highlighting the selective pressure on the pathogen to accommodate OsSWEET14 polymorphism, and reciprocally the apparent limited possibilities for the host to create variability at this particular S gene. Analysis of xa41(t) conservation across the Oryza genus enabled us to hypothesize scenarios as to its evolutionary history, prior to and during domestication. Our findings demonstrate that resistance through TAL effector-dependent loss of S-gene expression can be greatly fostered upon knowledge-based molecular screening of a large collection of host plants.© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.


September 22, 2019  |  

Molecular characterization of eukaryotic algal communities in the tropical phyllosphere based on real-time sequencing of the 18S rDNA gene.

Foliicolous algae are a common occurrence in tropical forests. They are referable to a few simple morphotypes (unicellular, sarcinoid-like or filamentous), which makes their morphology of limited usefulness for taxonomic studies and species diversity assessments. The relationship between algal community and their host phyllosphere was not clear. In order to obtain a more accurate assessment, we used single molecule real-time sequencing of the 18S rDNA gene to characterize the eukaryotic algal community in an area of South-western China.We annotated 2922 OTUs belonging to five classes, Ulvophyceae, Trebouxiophyceae, Chlorophyceae, Dinophyceae and Eustigmatophyceae. Novel clades formed by large numbers sequences of green algae were detected in the order Trentepohliales (Ulvophyceae) and the Watanabea clade (Trebouxiophyceae), suggesting that these foliicolous communities may be substantially more diverse than so far appreciated and require further research. Species in Trentepohliales, Watanabea clade and Apatococcus clade were detected as the core members in the phyllosphere community studied. Communities from different host trees and sampling sites were not significantly different in terms of OTUs composition. However, the communities of Musa and Ravenala differed from other host plants significantly at the genus level, since they were dominated by Trebouxiophycean epiphytes.The cryptic diversity of eukaryotic algae especially Chlorophytes in tropical phyllosphere is very high. The community structure at species-level has no significant relationship either with host phyllosphere or locations. The core algal community in tropical phyllopshere is consisted of members from Trentepohliales, Watanabea clade and Apatococcus clade. Our study provided a large amount of novel 18S rDNA sequences that will be useful to unravel the cryptic diversity of phyllosphere eukaryotic algae and for comparisons with similar future studies on this type of communities.


September 22, 2019  |  

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The de novo assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5′ and 3′ untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.


September 22, 2019  |  

Limited effects of variable-retention harvesting on fungal communities decomposing fine roots in coastal temperate rainforests.

Fine root litter is the principal source of carbon stored in forest soils and a dominant source of carbon for fungal decomposers. Differences in decomposer capacity between fungal species may be important determinants of fine-root decomposition rates. Variable-retention harvesting (VRH) provides refuge for ectomycorrhizal fungi, but its influence on fine-root decomposers is unknown, as are the effects of functional shifts in these fungal communities on carbon cycling. We compared fungal communities decomposing fine roots (in litter bags) under VRH, clear-cut, and uncut stands at two sites (6 and 13 years postharvest) and two decay stages (43 days and 1 year after burial) in Douglas fir forests in coastal British Columbia, Canada. Fungal species and guilds were identified from decomposed fine roots using high-throughput sequencing. Variable retention had short-term effects on ß-diversity; harvest treatment modified the fungal community composition at the 6-year-postharvest site, but not at the 13-year-postharvest site. Ericoid and ectomycorrhizal guilds were not more abundant under VRH, but stand age significantly structured species composition. Guild composition varied by decay stage, with ruderal species later replaced by saprotrophs and ectomycorrhizae. Ectomycorrhizal abundance on decomposing fine roots may partially explain why fine roots typically decompose more slowly than surface litter. Our results indicate that stand age structures fine-root decomposers but that decay stage is more important in structuring the fungal community than shifts caused by harvesting. The rapid postharvest recovery of fungal communities decomposing fine roots suggests resiliency within this community, at least in these young regenerating stands in coastal British Columbia.IMPORTANCE Globally, fine roots are a dominant source of carbon in forest soils, yet the fungi that decompose this material and that drive the sequestration or respiration of this carbon remain largely uncharacterized. Fungi vary in their capacity to decompose plant litter, suggesting that fungal community composition is an important determinant of decomposition rates. Variable-retention harvesting is a forestry practice that modifies fungal communities by providing refuge for ectomycorrhizal fungi. We evaluated the effects of variable retention and clear-cut harvesting on fungal communities decomposing fine roots at two sites (6 and 13 years postharvest), at two decay stages (43 days and 1 year), and in uncut stands in temperate rainforests. Harvesting impacts on fungal community composition were detected only after 6 years after harvest. We suggest that fungal community composition may be an important factor that reduces fine-root decomposition rates relative to those of above-ground plant litter, which has important consequences for forest carbon cycling. Copyright © 2018 American Society for Microbiology.


September 22, 2019  |  

Community profiling of Fusarium in combination with other plant associated fungi in different crop species using SMRT Sequencing.

Fusarium head blight, caused by fungi from the genus Fusarium, is one of the most harmful cereal diseases, resulting not only in severe yield losses but also in mycotoxin contaminated and health-threatening grains. Fusarium head blight is caused by a diverse set of species that have different host ranges, mycotoxin profiles and responses to agricultural practices. Thus, understanding the composition of Fusarium communities in the field is crucial for estimating their impact and also for the development of effective control measures. Up to now, most molecular tools that monitor Fusarium communities on plants are limited to certain species and do not distinguish other plant associated fungi. To close these gaps, we developed a sequencing-based community profiling methodology for crop-associated fungi with a focus on the genus Fusarium. By analyzing a 1600 bp long amplicon spanning the highly variable segments ITS and D1-D3 of the ribosomal operon by PacBio SMRT sequencing, we were able to robustly quantify Fusarium down to species level through clustering against reference sequences. The newly developed methodology was successfully validated in mock communities and provided similar results as the culture-based assessment of Fusarium communities by seed health tests in grain samples from different crop species. Finally, we exemplified the newly developed methodology in a field experiment with a wheat-maize crop sequence under different cover crop and tillage regimes. We analyzed wheat straw residues, cover crop shoots and maize grains and we could reveal that the cover crop hairy vetch (Vicia villosa) acts as a potent alternative host for Fusarium (OTU F.ave/tri) showing an eightfold higher relative abundance compared with other cover crop treatments. Moreover, as the newly developed methodology also allows to trace other crop-associated fungi, we found that vetch and green fallow hosted further fungal plant pathogens including Zymoseptoria tritici. Thus, besides their beneficial traits, cover crops can also entail phytopathological risks by acting as alternative hosts for Fusarium and other noxious plant pathogens. The newly developed sequencing based methodology is a powerful diagnostic tool to trace Fusarium in combination with other fungi associated to different crop species.


September 22, 2019  |  

Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits.

Diversity of microbial organisms is linked to global climatic gradients. The genus Phytophthora includes both aquatic and terrestrial plant pathogenic species that display a large variation of functional traits. The extent to which the physical environment (water or soil) modulates the interaction of microorganisms with climate is unknown. Here, we explored the main environmental drivers of diversity and functional trait composition of Phytophthora communities. Communities were obtained by a novel metabarcoding setup based on PacBio sequencing of river filtrates in 96 river sites along a geographical gradient. Species were classified as terrestrial or aquatic based on their phylogenetic clade. Overall, terrestrial and aquatic species showed contrasting patterns of diversity. For terrestrial species, precipitation was a stronger driver than temperature, and diversity and functional diversity decreased with decreasing temperature and precipitation. In cold and dry areas, the dominant species formed resistant structures and had a low optimum temperature. By contrast, for aquatic species, temperature and water chemistry were the strongest drivers, and diversity increased with decreasing temperature and precipitation. Within the same area, environmental filtering affected terrestrial species more strongly than aquatic species (20% versus 3% of the studied communities, respectively). Our results highlight the importance of functional traits and the physical environment in which microorganisms develop their life cycle when predicting their distribution under changing climatic conditions. Temperature and rainfall may be buffered differently by water and soil, and thus pose contrasting constrains to microbial assemblies.


September 22, 2019  |  

Long-term microbiota and virome in a Zürich patient after fecal transplantation against Clostridium difficile infection.

Fecal microbiota transplantation (FMT) is an emerging therapeutic option for Clostridium difficile infections that are refractory to conventional treatment. FMT introduces fecal microbes into the patient’s intestine that prevent the recurrence of C. difficile, leading to rapid expansion of bacteria characteristic of healthy microbiota. However, the long-term effects of FMT remain largely unknown. The C. difficile patient described in this paper revealed protracted microbiota adaptation processes from 6 to 42 months post-FMT. Ultimately, bacterial communities were donor similar, suggesting sustainable stool engraftment. Since little is known about the consequences of transmitted viruses during C. difficile infection, we also interrogated virome changes. Our approach allowed identification of about 10 phage types per sample that represented larger viral communities, and phages were found to be equally abundant in the cured patient and donor. The healthy microbiota appears to be characterized by low phage abundance. Although viruses were likely transferred, the patient established a virome distinct from the donor. Surprisingly, the patient had sequences of algal giant viruses (chloroviruses) that have not previously been reported for the human gut. Chloroviruses have not been associated with intestinal disease, but their presence in the oropharynx may influence cognitive abilities. The findings suggest that the virome is an important indicator of health or disease. A better understanding of the role of viruses in the gut ecosystem may uncover novel microbiota-modulating therapeutic strategies.© 2016 New York Academy of Sciences.


September 22, 2019  |  

Impacts of experimentally accelerated forest succession on belowground plant and fungal communities

Understanding how soil processes, belowground plant and fungal species composition, and nutrient cycles are altered by disturbances is essential for understanding the role forests play in mitigating global climate change. Here we ask: How are root and fungal communities altered in a mid-successional forest during shifts in dominant tree species composition? This study utilizes the Forest Accelerated Succession ExperimenT (FASET) at the University of Michigan Biological Station (UMBS) as a platform for addressing this question. FASET consists of a 39-ha treatment in which all mature early successional aspen (Populus spp.) and paper birch (Betula papyrifera) were killed by stem-girdling in 2008. Four years after girdling, neither overall fungal diversity indices, plant diversity indices, nor root biomass differed between girdled (treated) and non-girdled (reference) stands. However, experimental advancement of succession by removal of aspen and birch resulted in 1) a shift in fungal functional groups, with significantly less ectomycorrhizal fungi, 2) a trend toward less arbuscular mycorrhizal fungi, and 3) a significant increase in the proportion of saprotrophs in girdled stands. In addition to shifts in functional groups between treated and untreated stands, ectomycorrhizal fungi proportions were negatively correlated with NH4+ and total dissolved inorganic nitrogen (DIN) in soil. This research illustrates the propensity for disturbances in forest ecosystems to shift fungal community composition, which has implications for carbon storage and nutrient cycling in soils under future climate scenarios.


September 22, 2019  |  

Characterization of the Rosellinia necatrix transcriptome and genes related to pathogenesis by single-molecule mRNA sequencing.

White root rot disease, caused by the pathogen Rosellinia necatrix, is one of the world’s most devastating plant fungal diseases and affects several commercially important species of fruit trees and crops. Recent global outbreaks of R. necatrix and advances in molecular techniques have both increased interest in this pathogen. However, the lack of information regarding the genomic structure and transcriptome of R. necatrix has been a barrier to the progress of functional genomic research and the control of this harmful pathogen. Here, we identified 10,616 novel full-length transcripts from the filamentous hyphal tissue of R. necatrix (KACC 40445 strain) using PacBio single-molecule sequencing technology. After annotation of the unigene sets, we selected 14 cell cycle-related genes, which are likely either positively or negatively involved in hyphal growth by cell cycle control. The expression of the selected genes was further compared between two strains that displayed different growth rates on nutritional media. Furthermore, we predicted pathogen-related effector genes and cell wall-degrading enzymes from the annotated gene sets. These results provide the most comprehensive transcriptomal resources for R. necatrix, and could facilitate functional genomics and further analyses of this important phytopathogen.


September 22, 2019  |  

Assessing the gene content of the megagenome: sugar pine (Pinus lambertiana).

Sugar pine (Pinus lambertiana Douglas) is within the subgenus Strobus with an estimated genome size of 31 Gbp. Transcriptomic resources are of particular interest in conifers due to the challenges presented in their megagenomes for gene identification. In this study, we present the first comprehensive survey of the P. lambertiana transcriptome through deep sequencing of a variety of tissue types to generate more than 2.5 billion short reads. Third generation, long reads generated through PacBio Iso-Seq has been included for the first time in conifers to combat the challenges associated with de novo transcriptome assembly. A technology comparison is provided here contribute to the otherwise scarce comparisons of 2nd and 3rd generation transcriptome sequencing approaches in plant species. In addition, the transcriptome reference was essential for gene model identification and quality assessment in the parallel project responsible for sequencing and assembly of the entire genome. In this study, the transcriptomic data was also used to address some of the questions surrounding lineage-specific Dicer-like proteins in conifers. These proteins play a role in the control of transposable element proliferation and the related genome expansion in conifers. Copyright © 2016 Author et al.


September 22, 2019  |  

Single-cell (meta-)genomics of a dimorphic Candidatus Thiomargarita nelsonii reveals genomic plasticity.

The genus Thiomargarita includes the world’s largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group I intron also carried a MITE sequence that, like the hupL MITE family, occurs broadly across the genome. The presence of a high degree of mobile elements in genes central to Thiomargarita’s core metabolism has not been previously reported in free-living bacteria and suggests a highly mutable genome.


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