April 21, 2020  |  

Complete genome of Pseudomonas sp. DMSP-1 isolated from the Arctic seawater of Kongsfjorden, Svalbard

The genus Pseudomonas is highly metabolically diverse and has colonized a wide range of ecological niches. The strain Pseudomonas sp. DMSP-1 was isolated from Arctic seawater (Kongsfjorden, Svalbard) using dimethylsulfoniopropionate (DMSP) as the sole carbon source. To better understand its role in the Arctic coastal ecosystem, the genome of Pseudomonas sp. strain DMSP-1 was completely sequenced. The genome contained a circular chromosome of 6,282,445?bp with an average GC content of 60.01?mol%. A total of 5510 protein coding genes, 70 tRNA genes and 19 rRNA genes were obtained. However, no genes encoding known enzymes associated with DMSP catabolism were identified in the genome, suggesting that novel DMSP degradation genes might exist in Pseudomonas sp. strain DMSP-1.


April 21, 2020  |  

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Whole Genome Sequencing and Analysis of Chlorimuron-Ethyl Degrading Bacteria Klebsiella pneumoniae 2N3.

Klebsiella pneumoniae 2N3 is a strain of gram-negative bacteria that can degrade chlorimuron-ethyl and grow with chlorimuron-ethyl as the sole nitrogen source. The complete genome of Klebsiella pneumoniae 2N3 was sequenced using third generation high-throughput DNA sequencing technology. The genomic size of strain 2N3 was 5.32 Mb with a GC content of 57.33% and a total of 5156 coding genes and 112 non-coding RNAs predicted. Two hydrolases expressed by open reading frames (ORFs) 0934 and 0492 were predicted and experimentally confirmed by gene knockout to be involved in the degradation of chlorimuron-ethyl. Strains of ?ORF 0934, ?ORF 0492, and wild type (WT) reached their highest growth rates after 8-10 hours in incubation. The degradation rates of chlorimuron-ethyl by both ?ORF 0934 and ?ORF 0492 decreased in comparison to the WT during the first 8 hours in culture by 25.60% and 24.74%, respectively, while strains ?ORF 0934, ?ORF 0492, and the WT reached the highest degradation rates of chlorimuron-ethyl in 36 hours of 74.56%, 90.53%, and 95.06%, respectively. This study provides scientific evidence to support the application of Klebsiella pneumoniae 2N3 in bioremediation to control environmental pollution.


April 21, 2020  |  

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81?kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR The length of the target site was 52 or 55?bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Complete Sequence of a Novel Multidrug-Resistant Pseudomonas putida Strain Carrying Two Copies of qnrVC6.

This study aimed at identification and characterization of a novel multidrug-resistant Pseudomonas putida strain Guangzhou-Ppu420 carrying two copies of qnrVC6 isolated from a hospital in Guangzhou, China, in 2012. Antimicrobial susceptibility was tested by Vitek2™ Automated Susceptibility System and Etest™ strips, and whole-genome sequencing facilitated analysis of its multidrug resistance. The genome has a length of 6,031,212?bp and an average G?+?C content of 62.01%. A total of 5,421 open reading frames were identified, including eight 5S rRNA, seven 16S rRNA, and seven 23S rRNA, and 76 tRNA genes. Importantly, two copies of qnrVC6 gene with three ISCR1 around, a blaVIM-2 carrying integron In528, a novel gcu173 carrying integron In1348, and six antibiotic resistance genes were identified. This is the first identification of two copies of the qnrVC6 gene in a single P. putida isolate and a class 1 integron In1348.


April 21, 2020  |  

Complete genome of Pseudoalteromonas atlantica ECSMB14104, a Gammaproteobacterium inducing mussel settlement

Pseudoalteromonas is widely distributed in the marine environments and the biofilms formed by Pseudoalteromonas promote settlement of many species of invertebrates. Here, we show the complete genome of Pseudoalteromonas atlantica ECSMB14104, which was isolated from biofilms formed in the East China Sea and exhibited inducing activity on the Mytilus coruscus settlement. Complete genome of this strain containsa total of 3325 genes and the GC content of 41.02%. This genomic information is contributed to molecular mechanism of P. atlantica ECSMB14104 regulating mussel settlement.


April 21, 2020  |  

Complete genome sequence of Pseudomonas frederiksbergensis ERDD5:01 revealed genetic bases for survivability at high altitude ecosystem and bioprospection potential.

Pseudomonas frederiksbergensis ERDD5:01 is a psychrotrophic bacteria isolated from the glacial stream flowing from East Rathong glacier in Sikkim Himalaya. The strain showed survivability at high altitude stress conditions like freezing, frequent freeze-thaw cycles, and UV-C radiations. The complete genome of 5,746,824?bp circular chromosome and a plasmid of 371,027?bp was sequenced to understand the genetic basis of its survival strategy. Multiple copies of cold-associated genes encoding cold active chaperons, general stress response, osmotic stress, oxidative stress, membrane/cell wall alteration, carbon storage/starvation and, DNA repair mechanisms supported its survivability at extreme cold and radiations corroborating with the bacterial physiological findings. The molecular cold adaptation analysis in comparison with the genome of 15 mesophilic Pseudomonas species revealed functional insight into the strategies of cold adaptation. The genomic data also revealed the presence of industrially important enzymes.Copyright © 2018 Elsevier Inc. All rights reserved.


April 21, 2020  |  

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.


April 21, 2020  |  

Characterization of the genome of a Nocardia strain isolated from soils in the Qinghai-Tibetan Plateau that specifically degrades crude oil and of this biodegradation.

A strain of Nocardia isolated from crude oil-contaminated soils in the Qinghai-Tibetan Plateau degrades nearly all components of crude oil. This strain was identified as Nocardia soli Y48, and its growth conditions were determined. Complete genome sequencing showed that N. soli Y48 has a 7.3?Mb genome and many genes responsible for hydrocarbon degradation, biosurfactant synthesis, emulsification and other hydrocarbon degradation-related metabolisms. Analysis of the clusters of orthologous groups (COGs) and genomic islands (GIs) revealed that Y48 has undergone significant gene transfer events to adapt to changing environmental conditions (crude oil contamination). The structural features of the genome might provide a competitive edge for the survival of N. soli Y48 in oil-polluted environments and reflect the adaptation of coexisting bacteria to distinct nutritional niches.Copyright © 2018. Published by Elsevier Inc.


April 21, 2020  |  

The complete genome of the antifungal bacterium Pseudomonas sp. strain MS82

The genomic sequence of Pseudomonas sp. strain MS82 isolated from the rhizosphere of a soybean plant is reported and analyzed in relation to its extensive antifungal activity. Broth media used for production of the antifungal extract from strain MS82 against the mushroom pathogen Trichoderma viride were optimized using the routine plate bioassays. Culture extract of strain 82 in the peptone-K2HPO4-MgSO4 medium (PKM; peptone 20 g/L, K2HPO4 1.5 g/L, MgSO4 1.5 g/L and sterilized water) showed the best antifungal activity with an inhibition rate of 88.69thinspacetextpmthinspace3.87% to the fungal pathogen. Control efficacy of the T. viride contamination was investigated in mushroom production compost. The disease severity index of P. ostreatus hyphae infected by T. viride of treatment mixed with MS82 supernatant (38.33thinspacetextpmthinspace5.20%) was lower than that of the compost mixed with non-inoculated broth (97.50thinspacetextpmthinspace2.50%). The multilocus sequence analysis, containing four partial sequences from the gyrB, rpoB, recA and rpoD, suggests that strain MS82 is a Pseudomonas strain. The strain MS82 genome consists of a circular chromosome of 6,207,556 bp that was predicted to encode 5401 proteins and 131 RNA genes. Genome analysis revealed the presence of the gene clusters for biosynthesis of antifungal compounds, such as phenazine, pyocyanin, pyoverdine, volatile HCN and cyclic lipopeptides (arthrofactin). Genome analysis presented in the report will provide insights into development of biological control for fungal contamination in mushroom cultivation.


April 21, 2020  |  

Comparative genome analysis provides novel insight into the interaction of Aquimarina sp. AD1, BL5 and AD10 with their macroalgal host.

The Aquimarina genus is widely distributed throughout the marine environment, however little is understood regarding its ecological role, particularly when in association with eukaryotic hosts. Here, we examine the genomes of two opportunistic pathogens, Aquimarina sp. AD1 and BL5, and a non-pathogenic strain Aquimarina sp. AD10, that were isolated from diseased individuals of the red alga Delisea pulchra. Each strain encodes multiple genes for the degradation of marine carbohydrates and vitamin biosynthesis. These traits are hypothesised to promote nutrient exchange between the Aquimarina strains and their algal host, facilitating a close symbiotic relationship. Moreover, each strain harbours the necessary genes for the assembly of a Type 9 Secretion System (T9SS) and the associated gliding motility apparatus. In addition to these common features, pathogenic strains AD1 and BL5, encode genes for the production of flexirubin type pigments and a number of unique non-ribosomal peptide synthesis (NRPS) gene clusters, suggesting a role for these uncharacterised traits in virulence. This study provides valuable insight into the potential ecological role of Aquimarina in the marine environment and the complex factors driving pathogenesis and symbiosis in this genus.Copyright © 2019 Elsevier B.V. All rights reserved.


April 21, 2020  |  

In-Depth Genomic and Phenotypic Characterization of the Antarctic Psychrotolerant Strain Pseudomonas sp. MPC6 Reveals Unique Metabolic Features, Plasticity, and Biotechnological Potential.

We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments.


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