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September 22, 2019

Acquisition of resistance to carbapenem and macrolide-mediated quorum sensing inhibition by Pseudomonas aeruginosa via ICE Tn4371 6385

Pseudomonas aeruginosa can cause life-threatening infections in immunocompromised patients. The first-line agents to treat P. aeruginosa infections are carbapenems. However, the emergence of carbapenem-resistant P. aeruginosa strains greatly compromised the effec- tiveness of carbapenem treatment, which makes the surveillance on their spreading and transmission important. Here we characterized the full-length genomes of two carbapenem- resistant P. aeruginosa clinical isolates that are capable of producing New Delhi metallo-ß- lactamase-1 (NDM-1). We show that blaNDM-1 is carried by a novel integrative and conjugative element (ICE) ICETn43716385, which also carries the macrolide resistance gene msr(E) and the florfenicol resistance gene floR. By exogenously expressing msr(E) in P. aeruginosa laboratory strains, we show that Msr(E) can abolish azithromycin-mediated quorum sensing inhibition in vitro and anti-Pseudomonas effect in vivo. We conclude that ICEs are important in transmitting carbapenem resistance, and that anti-virulence treatment of P. aeruginosa infections using sub-inhibitory concentrations of macrolides can be challenged by horizontal gene transfer.

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September 22, 2019

Genome-based evolutionary history of Pseudomonas spp.

Pseudomonas is a large and diverse genus of Gammaproteobacteria. To provide a framework for discovery of evolutionary and taxonomic relationships of these bacteria, we compared the genomes of type strains of 163 species and 3 additional subspecies of Pseudomonas, including 118 genomes sequenced herein. A maximum likelihood phylogeny of the 166 type strains based on protein sequences of 100 single-copy orthologous genes revealed thirteen groups of Pseudomonas, composed of two to sixty three species each. Pairwise average nucleotide identities and alignment fractions were calculated for the data set of the 166 type strains and 1224 genomes of Pseudomonas available in public databases. Results revealed that 394 of the 1224 genomes were distinct from any type strain, suggesting that the type strains represent only a fraction of the genomic diversity of the genus. The core genome of Pseudomonas was determined to contain 794 genes conferring primarily housekeeping functions. The results of this study provide a phylogenetic framework for future studies aiming to resolve the classification and phylogenetic relationships, identify new gene functions and phenotypes, and explore the ecological and metabolic potential of the Pseudomonas spp.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Insect symbionts as valuable grist for the biotechnological mill: an alkaliphilic silkworm gut bacterium for efficient lactic acid production.

Insects constitute the most abundant and diverse animal class and act as hosts to an extraordinary variety of symbiotic microorganisms. These microbes living inside the insects play critical roles in host biology and are also valuable bioresources. Enterococcus mundtii EMB156, isolated from the larval gut (gut pH >10) of the model organism Bombyx mori (Lepidoptera: Bombycidae), efficiently produces lactic acid, an important metabolite for industrial production of bioplastic materials. E. mundtii EMB156 grows well under alkaline conditions and stably converts various carbon sources into lactic acid, offering advantages in downstream fermentative processes. High-yield lactic acid production can be achieved by the strain EMB156 from renewable biomass substrates under alkaline pretreatments. Single-molecule real-time (SMRT) sequencing technology revealed its 3.01 Mbp whole genome sequence. A total of 2956 protein-coding sequences, 65 tRNA genes, and 6 rRNA operons were predicted in the EMB156 chromosome. Remarkable genomic features responsible for lactic acid fermentation included key enzymes involved in the pentose phosphate (PP)/glycolytic pathway, and an alpha amylase and xylose isomerase were characterized in EMB156. This genomic information coincides with the phenotype of E. mundtii EMB156, reflecting its metabolic flexibility in efficient lactate fermentation, and established a foundation for future biotechnological application. Interestingly, enzyme activities of amylase were quite stable in high-pH broths, indicating a possible mechanism for strong EMB156 growth in an alkaline environment, thereby facilitating lactic acid production. Together, these findings implied that valuable lactic acid-producing bacteria can be discovered efficiently by screening under the extremely alkaline conditions, as exemplified by gut microbial symbionts of Lepidoptera insects.

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September 22, 2019

Pm21 from Haynaldia villosa encodes a CC-NBS-LRR protein conferring powdery mildew resistance in wheat.

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive disease of wheat throughout the world. One of the most important environmental-friendly and economical methods to reduce wheat loss caused by Bgt is to develop highly resistant varieties (Kuraparthy et al., 2007). Pm21 from the wild species Haynaldia villosa (also known as Dasypyrum villosum) confers high resistance to Bgt in wheat throughout all growth stages. It has now become one of the most highly effective genetic loci introgressed into wheat from wild species, and the commercial varieties harboring Pm21 have been widely used in wheat production with more than 4 million hectares in China.

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September 22, 2019

Multiplex assessment of protein variant abundance by massively parallel sequencing.

Determining the pathogenicity of genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes requires generalizable, scalable assays. We describe variant abundance by massively parallel sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance simultaneously. We apply VAMP-seq to quantify the abundance of 7,801 single-amino-acid variants of PTEN and TPMT, proteins in which functional variants are clinically actionable. We identify 1,138 PTEN and 777 TPMT variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and show that p.Pro38Ser, which accounts for ~10% of PTEN missense variants in melanoma, functions via a dominant-negative mechanism. Finally, we demonstrate that VAMP-seq is applicable to other genes, highlighting its generalizability.

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September 22, 2019

Diversity and evolution of the emerging Pandoraviridae family.

With DNA genomes reaching 2.5?Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting pandoraviruses remained up to now the most complex viruses since their discovery in 2013. Our isolation of three new strains from distant locations and environments is now used to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses reveals many non-coding transcripts and significantly reduces the former set of predicted protein-coding genes. Here we show that the pandoraviruses exhibit an open pan-genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain-specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggest that de novo gene creation could contribute to the evolution of the giant pandoravirus genomes.

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September 22, 2019

Parallels between experimental and natural evolution of legume symbionts.

The emergence of symbiotic interactions has been studied using population genomics in nature and experimental evolution in the laboratory, but the parallels between these processes remain unknown. Here we compare the emergence of rhizobia after the horizontal transfer of a symbiotic plasmid in natural populations of Cupriavidus taiwanensis, over 10 MY ago, with the experimental evolution of symbiotic Ralstonia solanacearum for a few hundred generations. In spite of major differences in terms of time span, environment, genetic background, and phenotypic achievement, both processes resulted in rapid genetic diversification dominated by purifying selection. We observe no adaptation in the plasmid carrying the genes responsible for the ecological transition. Instead, adaptation was associated with positive selection in a set of genes that led to the co-option of the same quorum-sensing system in both processes. Our results provide evidence for similarities in experimental and natural evolutionary transitions and highlight the potential of comparisons between both processes to understand symbiogenesis.

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September 22, 2019

First report of the occurrence and whole-genome characterization of Edwardsiella tarda in the false killer whale (Pseudorca crassidens).

Although several Edwardsiella tarda infections have been reported, its pathogenic role in marine mammals has not been investigated at the genome level. We investigated the genome of E. tarda strain KC-Pc-HB1, isolated from the false killer whale (Pseudorca crassidens) found bycaught in South Korea. The obtained genome was similar to that of human pathogenic E. tarda strains, but distinct from other Edwardsiella species. Although type III and VI secretion systems, which are essential for the virulence of other Edwardsiella species, were absent, several virulence-related genes involved in the pathogenesis of E. tarda were found in the genome. These results provide important insights into the E. tarda infecting marine mammals and give valuable information on potential virulence factors in this pathogen.

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September 22, 2019

Landscape of the genome and host cell response of Mycobacterium shigaense reveals pathogenic features.

A systems approach was used to explore the genome and transcriptome of Mycobacterium shigaense, a new opportunistic pathogen isolated from a patient with a skin infection, and the host response transcriptome was assessed using a macrophage infection model. The M. shigaense genome comprises 5,207,883?bp, with 67.2% G+C content and 5098 predicted coding genes. Evolutionarily, the bacterium belongs to a cluster in the phylogenetic tree along with three target opportunistic pathogenic strains, namely, M. avium, M. triplex and M. simiae. Potential virulence genes are indeed expressed by M. shigaense under culture conditions. Phenotypically, M. shigaense had similar infection and replication capacities in a macrophage model as the opportunistic species compared to M. tuberculosis. M. shigaense activated NF-?B, TNF, cytokines and chemokines in the host innate immune-related signaling pathways and elicited an early response shared with pathogenic bacilli except M. tuberculosis. M. shigaense upregulated specific host response genes such as TLR7, CCL4 and CXCL5. We performed an integrated and comparative analysis of M. shigaense. Multigroup comparison indicated certain differences with typical pathogenic bacilli in terms of gene features and the macrophage response.

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September 22, 2019

A comprehensive understanding of the biocontrol potential of Bacillus velezensis LM2303 against Fusarium head blight.

Fusarium head blight (FHB) mainly caused by F. graminearum, always brings serious damage to wheat production worldwide. In this study, we found that strain LM2303 had strong antagonist activity against F. graminearum and significantly reduced disease severity of FHB with the control efficiency of 72.3% under field conditions. To gain a comprehensive understanding of the biocontrol potential of strain LM2303 against FHB, an integrated approach of genome mining and chemical analysis was employed. The whole genome of strain LM2303 was obtained and analyzed, showing the largest number of genes/gene clusters associated with biocontrol functions as compared with the known biocontrol strains (FZB42, M75, CAU B946). And strain LM2303 was accurately determined as a member of the B. velezensis clade using the phylogenomic analysis of single-copy core genes. Through genome mining, 13 biosynthetic gene clusters(BGCs) encoding secondary metabolites with biocontrol functions were identified, which were further confirmed through chemical analyses such as UHPLC-ESI-MS, including three antifungal metabolites (fengycin B, iturin A, and surfactin A), eight antibacterial metabolites (surfactin A, butirosin, plantazolicin and hydrolyzed plantazolicin, kijanimicin, bacilysin, difficidin, bacillaene A and bacillaene B, 7-o-malonyl macrolactin A and 7-o-succinyl macrolactin A), the siderophore bacillibactin, molybdenum cofactor and teichuronic acid. In addition, genes/gene clusters involved in plant colonization, plant growth promotion and induced systemic resistance were also found and analyzed, along with the corresponding metabolites. Finally, four different mechanisms of strain LM2303 involved in the biocontrol of FHB were putatively obtained. This work provides better insights into a mechanistic understanding of strain LM2303 in control of FHB, reinforcing the higher potential of this strain as a powerful biocontrol strain agent (BCA) for FHB control. The results also provide scientific reference and comparison for other biocontrol strains.

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September 22, 2019

Draft genome sequence of Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina, and Morchella septimelata.

Draft genomes of the species Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina and Morchella septimelata are presented. Both mating types (MAT1-1 and MAT1-2) of Cercospora beticola are included. Two strains of Coleophoma cylindrospora that produce sulfated homotyrosine echinocandin variants, FR209602, FR220897 and FR220899 are presented. The sequencing of Aspergillus mulundensis, Coleophoma cylindrospora and Phialophora cf. hyalina has enabled mapping of the gene clusters encoding the chemical diversity from the echinocandin pathways, providing data that reveals the complexity of secondary metabolism in these different species. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity (in some cases), biology and toxin production of these economically important fungi.

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September 22, 2019

Computational Modeling of Multidrug-Resistant Bacteria

Understanding how complex phenotypes arise from individual molecules and their interactions is a primary challenge in biology, and computational approaches have been increasingly employed to tackle this task. In this chapter, we describe current efforts by FIOCRUZ and partners to develop integrated computational models of multidrug-resistant bacteria. The bacterium chosen as the main focus of this effort is Pseudomonas aeruginosa, an opportunistic pathogen associated with a broad spectrum of infections in humans. Nowadays, P. aeruginosa is one of the main problems of healthcare-associated infections (HAI) in the world, because of its great capacity of survival in hospital environments and its intrinsic resistance to many antibiotics. Our overall research objective is to use integrated computational models to accurately predict a wide range of observable cellular behaviors of multidrug-resistant P. aeruginosa CCBH4851, which is a strain belonging to the clone ST277, endemic in Brazil. In this chapter, after a brief introduction to P. aeruginosa biology, we discuss the construction of metabolic and gene regulatory networks of P. aeruginosa CCBH 4851 from its genome. We also illustrate how these networks can be integrated into a single model, and we discuss methods for identifying potential therapeutic targets through integrated models.

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September 22, 2019

Co-culture of soil biofilm isolates enables the discovery of novel antibiotics

Bacterial natural products (NPs) are considered to be a promising source of drug discovery. However, the biosynthesis gene clusters (BGCs) of NP are not often expressed, making it difficult to identify them. Recently, the study of biofilm community showed bacteria may gain competitive advantages by the secretion of antibiotics, implying a possible way to screen antibiotic by evaluating the social behavior of bacteria. In this study, we have described an efficient workflow for novel antibiotic discovery by employing the bacterial social interaction strategy with biofilm cultivation, co-culture, transcriptomic and genomic methods. We showed that a biofilm dominant species, i.e. Pseudomonas sp. G7, which was isolated from cultivated soil biofilm community, was highly competitive in four-species biofilm communities, as the synergistic combinations preferred to exclude this strain while the antagonistic combinations did not. Through the analysis of transcriptomic changes in four-species co-culture and the complete genome of Pseudomonas sp. G7, we finally discovered two novel non-ribosomal polypeptide synthetic (NRPS) BGCs, whose products were predicted to have seven and six amino acid components, respectively. Furthermore, we provide evidence showing that only when Pseudomonas sp. G7 was co-cultivated with at least two or three other bacterial species can these BGC genes be induced, suggesting that the co-culture of the soil biofilm isolates is critical to the discovery of novel antibiotics. As a conclusion, we set a model of applying microbial interaction to the discovery of new antibiotics.

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September 22, 2019

Analysis of the complete genome sequence of Bacillus atrophaeus GQJK17 reveals its biocontrol characteristics as a plant growth-promoting rhizobacterium

Bacillus atrophaeus GQJK17 was isolated from the rhizosphere of Lycium barbarum L. in China, which was shown to be a plant growth-promoting rhizobacterium as a new biological agent against pathogenic fungi and gram-positive bacteria. We present its biological characteristics and complete genome sequence, which contains a 4,325,818 bp circular chromosome with 4,181 coding DNA sequences and a G+C content of 43.3%. A genome analysis revealed a total of 8 candidate gene clusters for producing antimicrobial secondary metabolites, including surfactin, bacillaene, fengycin, and bacillibactin. Some other antimicrobial and plant growth-promoting genes were also discovered. Our results provide insights into the genetic and biological basis of B. atrophaeus strains as a biocontrol agent for application in agriculture.

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September 22, 2019

De novo genome assembly of Oryza granulata reveals rapid genome expansion and adaptive evolution

The wild relatives of rice have adapted to different ecological environments and constitute a useful reservoir of agronomic traits for genetic improvement. Here we present the ~777?Mb de novo assembled genome sequence of Oryza granulata. Recent bursts of long-terminal repeat retrotransposons, especially RIRE2, led to a rapid twofold increase in genome size after O. granulata speciation. Universal centromeric tandem repeats are absent within its centromeres, while gypsy-type LTRs constitute the main centromere-specific repetitive elements. A total of 40,116 protein-coding genes were predicted in O. granulata, which is close to that of Oryza sativa. Both the copy number and function of genes involved in photosynthesis and energy production have undergone positive selection during the evolution of O. granulata, which might have facilitated its adaptation to the low light habitats. Together, our findings reveal the rapid genome expansion, distinctive centromere organization, and adaptive evolution of O. granulata.

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