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Asset Tag: Microbiology

April 21, 2020

Blast Fungal Genomes Show Frequent Chromosomal Changes, Gene Gains and Losses, and Effector Gene Turnover.

Pyricularia is a fungal genus comprising several pathogenic species causing the blast disease in monocots. Pyricularia oryzae, the best-known species, infects rice, wheat, finger millet, and other crops. As past comparative and population genomics studies mainly focused on isolates of P. oryzae, the genomes of the other Pyricularia species have not been well explored. In this study, we obtained a chromosomal-level genome assembly of the finger millet isolate P. oryzae MZ5-1-6 and also highly contiguous assemblies of Pyricularia sp. LS, P. grisea, and P. pennisetigena. The differences in the genomic content of repetitive DNA sequences could largely explain the variation in genome size among these new genomes. Moreover, we found extensive gene gains and losses and structural changes among Pyricularia genomes, including a large interchromosomal translocation. We searched for homologs of known blast effectors across fungal taxa and found that most avirulence effectors are specific to Pyricularia, whereas many other effectors share homologs with distant fungal taxa. In particular, we discovered a novel effector family with metalloprotease activity, distinct from the well-known AVR-Pita family. We predicted 751 gene families containing putative effectors in 7 Pyricularia genomes and found that 60 of them showed differential expression in the P. oryzae MZ5-1-6 transcriptomes obtained under experimental conditions mimicking the pathogen infection process. In summary, this study increased our understanding of the structural, functional, and evolutionary genomics of the blast pathogen and identified new potential effector genes, providing useful data for developing crops with durable resistance. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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April 21, 2020

Systematic evasion of the restriction-modification barrier in bacteria.

Bacteria that are recalcitrant to genetic manipulation using modern in vitro techniques are termed genetically intractable. Genetic intractability is a fundamental barrier to progress that hinders basic, synthetic, and translational microbiology research and development beyond a few model organisms. The most common underlying causes of genetic intractability are restriction-modification (RM) systems, ubiquitous defense mechanisms against xenogeneic DNA that hinder the use of genetic approaches in the vast majority of bacteria and exhibit strain-level variation. Here, we describe a systematic approach to overcome RM systems. Our approach was inspired by a simple hypothesis: if a synthetic piece of DNA lacks the highly specific target recognition motifs for a host’s RM systems, then it is invisible to these systems and will not be degraded during artificial transformation. Accordingly, in this process, we determine the genome and methylome of an individual bacterial strain and use this information to define the bacterium’s RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent “SyngenicDNA” tool, and propagate the tool as minicircle plasmids, termed SyMPL (SyngenicDNA Minicircle Plasmid) tools, before transformation. In a proof-of-principle of our approach, we demonstrate a profound improvement (five orders of magnitude) in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus This stealth-by-engineering SyngenicDNA approach is effective, flexible, and we expect in future applications could enable microbial genetics free of the restraints of restriction-modification barriers.Copyright © 2019 the Author(s). Published by PNAS.

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April 21, 2020

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-Hydroxytriazene Moiety.

Triacsins are a family of natural products having in common an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, their biosynthesis has remained unknown. Here we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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April 21, 2020

Metagenomic assembly through the lens of validation: recent advances in assessing and improving the quality of genomes assembled from metagenomes.

Metagenomic samples are snapshots of complex ecosystems at work. They comprise hundreds of known and unknown species, contain multiple strain variants and vary greatly within and across environments. Many microbes found in microbial communities are not easily grown in culture making their DNA sequence our only clue into their evolutionary history and biological function. Metagenomic assembly is a computational process aimed at reconstructing genes and genomes from metagenomic mixtures. Current methods have made significant strides in reconstructing DNA segments comprising operons, tandem gene arrays and syntenic blocks. Shorter, higher-throughput sequencing technologies have become the de facto standard in the field. Sequencers are now able to generate billions of short reads in only a few days. Multiple metagenomic assembly strategies, pipelines and assemblers have appeared in recent years. Owing to the inherent complexity of metagenome assembly, regardless of the assembly algorithm and sequencing method, metagenome assemblies contain errors. Recent developments in assembly validation tools have played a pivotal role in improving metagenomics assemblers. Here, we survey recent progress in the field of metagenomic assembly, provide an overview of key approaches for genomic and metagenomic assembly validation and demonstrate the insights that can be derived from assemblies through the use of assembly validation strategies. We also discuss the potential for impact of long-read technologies in metagenomics. We conclude with a discussion of future challenges and opportunities in the field of metagenomic assembly and validation. © The Author 2017. Published by Oxford University Press.

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April 21, 2020

Petunia-and Arabidopsis-Specific Root Microbiota Responses to Phosphate Supplementation

Phosphorus (P) is a limiting element for plant growth. Several root microbes, including arbuscular mycorrhizal fungi (AMF), have the capacity to improve plant nutrition and their abundance is known to depend on P fertility. However, how complex root-associated bacterial and fungal communities respond to various levels of P supplementation remains ill-defined. Here we investigated the responses of the root-associated bacteria and fungi to varying levels of P supply using 16S rRNA gene and internal transcribed spacer amplicon sequencing. We grew Petunia, which forms symbiosis with AMF, and the nonmycorrhizal model species Arabidopsis as a control in a soil that is limiting in plant-available P and we then supplemented the plants with complete fertilizer solutions that varied only in their phosphate concentrations. We searched for microbes, whose abundances varied by P fertilization, tested whether a core microbiota responding to the P treatments could be identified and asked whether bacterial and fungal co-occurrence patterns change in response to the varying P levels. Root microbiota composition varied substantially in response to the varying P application. A core microbiota was not identified as different bacterial and fungal groups responded to low-P conditions in Arabidopsis and Petunia. Microbes with P-dependent abundance patterns included Mortierellomycotina in Arabidopsis, while in Petunia, they included AMF and their symbiotic endobacteria. Of note, the P-dependent root colonization by AMF was reliably quantified by sequencing. The fact that the root microbiotas of the two plant species responded differently to low-P conditions suggests that plant species specificity would need to be considered for the eventual development of microbial products that improve plant P nutrition.

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April 21, 2020

Systematic Identification of Pathogenic Streptomyces sp. AMCC400023 That Causes Common Scab and Genomic Analysis of Its Pathogenicity Island.

Potato scab, a serious soilborne disease caused by Streptomyces spp., occurs in potato-growing areas worldwide and results in severe economic losses. In this paper, the pathogenicity of Streptomyces strain AMCC400023, isolated from potato scabs in Hebei Province, China, was verified systematically by the radish seedling test, the potato tuber slice assay, the potted back experiment, and the detection of phytotoxin thaxtomin A. Morphological, physiological, and biochemical characteristics were determined, and the 16S ribosomal RNA analyses of Streptomyces sp. AMCC400023 were carried out. To obtain the accurate taxonomic status of the pathogen strain, the whole genome was sequenced, and the phylogenetic tree among 31 Streptomyces genomes was formed. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) were analyzed, and at the same time, the toxicity-related genes between Streptomyces sp. AMCC400023 and Streptomyces scabiei were compared, all based on the whole-genome level. All of the data supported that, instead of a member of S. scabiei, test strain Streptomyces sp. AMCC400023 was a distinct phytopathogen of potato common scab, which had a relatively close relationship with S. scabiei while separating clearly from S. scabiei at least in the species level of taxonomic status. The complete pathogenicity island (PAI) composition of Streptomyces sp. AMCC400023 was identified, which contained a toxin region and a colonization region. It was conjectured that the PAI of Streptomyces sp. AMCC400023 might be directly or indirectly acquired from S. scabiei 87-22 by horizontal gene transfer, or at the very least, there was a very close homologous relationship between the two pathogens as indicated by a series of analyses, such as phylogenetic relationships among 31 Streptomyces species, ANI and isDDH analyses, PAI structure mapping, thaxtomin A synthetic gene cluster tree construction, and most important, the collinearity analysis at the genome level.

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April 21, 2020

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory. © 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020

Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi.

The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time1,2 and metabolic prowess3,4. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.

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April 21, 2020

Phenotypic and Genomic Analyses of Burkholderia stabilis Clinical Contamination, Switzerland.

A recent hospital outbreak related to premoistened gloves used to wash patients exposed the difficulties of defining Burkholderia species in clinical settings. The outbreak strain displayed key B. stabilis phenotypes, including the inability to grow at 42°C; we used whole-genome sequencing to confirm the pathogen was B. stabilis. The outbreak strain genome comprises 3 chromosomes and a plasmid, sharing an average nucleotide identity of 98.4% with B. stabilis ATCC27515 BAA-67, but with 13% novel coding sequences. The genome lacks identifiable virulence factors and has no apparent increase in encoded antimicrobial drug resistance, few insertion sequences, and few pseudogenes, suggesting this outbreak was an opportunistic infection by an environmental strain not adapted to human pathogenicity. The diversity among outbreak isolates (22 from patients and 16 from washing gloves) is only 6 single-nucleotide polymorphisms, although the genome remains plastic, with large elements stochastically lost from outbreak isolates.

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April 21, 2020

Investigation of the role of typhoid toxin in acute typhoid fever in a human challenge model.

Salmonella Typhi is a human host-restricted pathogen that is responsible for typhoid fever in approximately 10.9 million people annually1. The typhoid toxin is postulated to have a central role in disease pathogenesis, the establishment of chronic infection and human host restriction2-6. However, its precise role in typhoid disease in humans is not fully defined. We studied the role of typhoid toxin in acute infection using a randomized, double-blind S. Typhi human challenge model7. Forty healthy volunteers were randomized (1:1) to oral challenge with 104 colony-forming units of wild-type or an isogenic typhoid toxin deletion mutant (TN) of S. Typhi. We observed no significant difference in the rate of typhoid infection (fever =38?°C for =12?h and/or S. Typhi bacteremia) between participants challenged with wild-type or TN S. Typhi (15 out of 21 (71%) versus 15 out of 19 (79%); P?=?0.58). The duration of bacteremia was significantly longer in participants challenged with the TN strain compared with wild-type (47.6 hours (28.9-97.0) versus 30.3(3.6-49.4); P = 0.001). The clinical syndrome was otherwise indistinguishable between wild-type and TN groups. These data suggest that the typhoid toxin is not required for infection and the development of early typhoid fever symptoms within the context of a human challenge model. Further clinical data are required to assess the role of typhoid toxin in severe disease or the establishment of bacterial carriage.

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April 21, 2020

Paraburkholderia dokdonella sp. nov., isolated from a plant from the genus Campanula.

The novel Gram-stain-negative, rod-shaped, aerobic bacterial strain DCR-13T was isolated from a native plant belonging to the genus Campanula on Dokdo, an island in the Republic of Korea. Comparative analysis of the 16S rRNA gene sequence indicated that this strain is closely related to Paraburkholderia peleae PP52-1T (98.43% 16S rRNA gene sequence similarity), Paraburkholderia oxyphila NBRC 105797T (98.42%), Paraburkholderia sacchari IPT 101T (98.28%), Paraburkholderia mimosarum NBRC 106338T (97.80%), Paraburkholderia denitrificans KIS30-44T (97.46%), and Paraburkholderia paradise WAT (97.45%). This analysis of the 16S rRNA gene sequence also suggested that DCR-13T and the six closely related strains formed a clade within the genus Paraburkholderia, but that DCR-13T was clearly separated from the established species. DCR-13T had ubiquinone 8 as its predominant respiratory quinone, and its genomic DNA G + C content was 63.9 mol%. The isolated strain grew at a pH of 6.0-8.0 (with an optimal pH of 6.5), 0-4% w/v NaCl (with an optimal level of 0%), and a temperature of 18-42°C (with an optimal temperature of 30°C). The predominant fatty acids were C16:0, summed feature 8 (C18:1?7c/C18:1?6c), C17:0 cyclo, C19:0 cyclo ?8c, summed feature 3 (C16:1?6c/C16:1?7c) and summed feature 2 (C12:0 aldehyde), and the major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. On the basis of polyphasic evidence, it is proposed that strain DCR-13T (= KCTC 62811T = LMG 30889T) represents the type strain of a novel species, Paraburkholderia dokdonella sp. nov.

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April 21, 2020

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.

CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes  LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10?kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering. © 2019 Wiley Periodicals, Inc.

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April 21, 2020

Complete genome sequence and phylogenetic analysis of nosocomial pathogen Acinetobacter nosocomialis strain NCTC 8102.

Acinetobacter has emerged recently as one of the most challenging nosocomial pathogens because of its increased rate of antimicrobial resistance. The genetic complexity and genome diversity, as well as the lack of adequate knowledge on the pathogenic determinants of Acinetobacter strains often hinder with pathogenesis studies for the development of better therapeutics to tackle this nosocomial pathogen.In this study, we comparatively analyzed the whole genome sequence of a virulent Acinetobacternosocomialis strain NCTC 8102.The genomic DNA of A. nosocomialis NCTC 8102 was isolated and sequenced using PacBio RS II platform. The sequenced genome was functionally annotated and gene prediction was carried out using the program, Glimmer 3. The phylogenetic analysis of the genome was performed using Mega 6 program and the comparative genome analysis was carried out by BLAST (Basic Local Alignment Search Tool).The complete genome analysis depicted that the genome consists of a circular chromosome with an average G?+?C content of 38.7%. The genome comprises 3700 protein-coding genes, 96 RNA genes (18 rRNA, 74 tRNA and 4 ncRNA genes), and 91 pseudogenes. In addition, 6 prophage regions comprising 2 intact, 1 incomplete and 3 questionable ones and 18 genomic islands were identified in the genome, suggesting the possible occurrence of horizontal gene transfer in this strain. Comparative genome analysis of A. nosocomialis NCTC 8102 genome with the already sequenced A. nosocomialis strain SSA3 showed an average nucleotide identity of 99.0%. In addition, the number of prophages and genomic islands were higher in the A. nosocomialis NCTC 8102 genome compared to that of the strain SSA3. 14 of the genomic islands were unique to A. nosocomialis NCTC 8102 compared to strain SSA3 and they harbored genes which are involved in virulence, multidrug resistance, biofilm formation and bacterial pathogenesis.We sequenced the whole genome of A. nosocomialis strain NCTC 8102 followed by comparatively genome analysis. The study provides valuable information on the genetic features of A. nosocomialis strain and the data from this study would assist in further studies for the development of control measures for this nosocomial pathogen.

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April 21, 2020

Isolation, cloning and characterization of an azoreductase and the effect of salinity on its expression in a halophilic bacterium.

Understanding the molecular mechanisms of azo dye decolorization is important for the development of effective bioremediation for textile-colored wastewater. A halophilic bacterium Halomonas sp. strain GT was isolated, which could degrade the azo dye Acid Brilliant Scarlet GR at 10% NaCl. The complete genome sequence of this strain was obtained using the PacBio RS II platform. Genome annotation revealed that four proteins are related to decolorization of azo dyes, such as azoreductase, laccases, benzene 1,2-dioxygenase, and catechol 1,2-dioxygenase. The putative azoreductase gene of Halomonas sp. strain GT responsible for the decolorization of azo dye in high salt environment was isolated. Phylogenetic tree analysis showed that the azoG (azoreductase gene of Halomonas sp. strain GT) and its homologs constituted a new branch of the NADH depending azoreductases, with all the homologous sequence of the protein from halophilic bacteria. At high NaCl concentrations, azoreductase gene expression and azoreductase activity were restrained in Halomonas sp. strain GT, which resulted in low a decolorization rate. Copyright © 2018. Published by Elsevier B.V.

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April 21, 2020

Insights into the evolution and drug susceptibility of Babesia duncani from the sequence of its mitochondrial and apicoplast genomes.

Babesia microti and Babesia duncani are the main causative agents of human babesiosis in the United States. While significant knowledge about B. microti has been gained over the past few years, nothing is known about B. duncani biology, pathogenesis, mode of transmission or sensitivity to currently recommended therapies. Studies in immunocompetent wild type mice and hamsters have shown that unlike B. microti, infection with B. duncani results in severe pathology and ultimately death. The parasite factors involved in B. duncani virulence remain unknown. Here we report the first known completed sequence and annotation of the apicoplast and mitochondrial genomes of B. duncani. We found that the apicoplast genome of this parasite consists of a 34?kb monocistronic circular molecule encoding functions that are important for apicoplast gene transcription as well as translation and maturation of the organelle’s proteins. The mitochondrial genome of B. duncani consists of a 5.9?kb monocistronic linear molecule with two inverted repeats of 48?bp at both ends. Using the conserved cytochrome b (Cytb) and cytochrome c oxidase subunit I (coxI) proteins encoded by the mitochondrial genome, phylogenetic analysis revealed that B. duncani defines a new lineage among apicomplexan parasites distinct from B. microti, Babesia bovis, Theileria spp. and Plasmodium spp. Annotation of the apicoplast and mitochondrial genomes of B. duncani identified targets for development of effective therapies. Our studies set the stage for evaluation of the efficacy of these drugs alone or in combination against B. duncani in culture as well as in animal models.Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

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