Menu
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.


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.


April 21, 2020  |  

The complete genome sequence of Ethanoligenens harbinense reveals the metabolic pathway of acetate-ethanol fermentation: A novel understanding of the principles of anaerobic biotechnology.

Ethanol-type fermentation is one of three main fermentation types in the acidogenesis of anaerobic treatment systems. Non-spore-forming Ethanoligenens is as a typical genus capable of ethanol-type fermentation in mixed culture (i.e. acetate-ethanol fermentation). This genus can produce ethanol, acetate, CO2, and H2 using carbohydrates, and has application potential in anaerobic bioprocesses. Here, the complete genome sequences and methylome of Ethanoligenens harbinense strains with different autoaggregative and coaggregative abilities were obtained using the PacBio single-molecule real-time sequencing platform. The genome size of E. harbinense strains was about 2.97-3.10?Mb with 55.5% G+C content. 3020-3153 genes were annotated, most of which were methylated at specific sites or motifs. The methylation types included 6mA, 4mC, and unknown types. Comparative genomic analysis demonstrated low levels of genetic similarity between E. harbinense and other well-known hydrogen-producing bacteria (i.e., Clostridium and Thermoanaerobacter) in phylogenesis. Hydrogen production of E. harbinense was catalyzed by genes that encode [FeFe]-hydrogenases and that were synthesized by three maturases of [FeFe]-H2ase. The metabolic mechanism of H2-ethanol co-production fermentation, catalyzed by pyruvate ferredoxin oxidoreductase was proposed. This study provides genetic and evolutionary information of a model genus for the further investigation of the metabolic pathway and regulatory network of ethanol-type fermentation and anaerobic bioprocesses for waste or wastewater treatment.Copyright © 2019. Published by Elsevier Ltd.


April 21, 2020  |  

Complete Assembly of the Genome of an Acidovorax citrulli Strain Reveals a Naturally Occurring Plasmid in This Species.

Acidovorax citrulli is the causal agent of bacterial fruit blotch (BFB), a serious threat to cucurbit crop production worldwide. Based on genetic and phenotypic properties, A. citrulli strains are divided into two major groups: group I strains have been generally isolated from melon and other non-watermelon cucurbits, while group II strains are closely associated with watermelon. In a previous study, we reported the genome of the group I model strain, M6. At that time, the M6 genome was sequenced by MiSeq Illumina technology, with reads assembled into 139 contigs. Here, we report the assembly of the M6 genome following sequencing with PacBio technology. This approach not only allowed full assembly of the M6 genome, but it also revealed the occurrence of a ~53 kb plasmid. The M6 plasmid, named pACM6, was further confirmed by plasmid extraction, Southern-blot analysis of restricted fragments and obtention of M6-derivative cured strains. pACM6 occurs at low copy numbers (average of ~4.1 ± 1.3 chromosome equivalents) in A. citrulli M6 and contains 63 open reading frames (ORFs), most of which (55.6%) encoding hypothetical proteins. The plasmid contains several genes encoding type IV secretion components, and typical plasmid-borne genes involved in plasmid maintenance, replication and transfer. The plasmid also carries an operon encoding homologs of a Fic-VbhA toxin-antitoxin (TA) module. Transcriptome data from A. citrulli M6 revealed that, under the tested conditions, the genes encoding the components of this TA system are among the highest expressed genes in pACM6. Whether this TA module plays a role in pACM6 maintenance is still to be determined. Leaf infiltration and seed transmission assays revealed that, under tested conditions, the loss of pACM6 did not affect the virulence of A. citrulli M6. We also show that pACM6 or similar plasmids are present in several group I strains, but absent in all tested group II strains of A. citrulli.


April 21, 2020  |  

Sequence properties of certain GC rich avian genes, their origins and absence from genome assemblies: case studies.

More and more eukaryotic genomes are sequenced and assembled, most of them presented as a complete model in which missing chromosomal regions are filled by Ns and where a few chromosomes may be lacking. Avian genomes often contain sequences with high GC content, which has been hypothesized to be at the origin of many missing sequences in these genomes. We investigated features of these missing sequences to discover why some may not have been integrated into genomic libraries and/or sequenced.The sequences of five red jungle fowl cDNA models with high GC content were used as queries to search publicly available datasets of Illumina and Pacbio sequencing reads. These were used to reconstruct the leptin, TNFa, MRPL52, PCP2 and PET100 genes, all of which are absent from the red jungle fowl genome model. These gene sequences displayed elevated GC contents, had intron sizes that were sometimes larger than non-avian orthologues, and had non-coding regions that contained numerous tandem and inverted repeat sequences with motifs able to assemble into stable G-quadruplexes and intrastrand dyadic structures. Our results suggest that Illumina technology was unable to sequence the non-coding regions of these genes. On the other hand, PacBio technology was able to sequence these regions, but with dramatically lower efficiency than would typically be expected.High GC content was not the principal reason why numerous GC-rich regions of avian genomes are missing from genome assembly models. Instead, it is the presence of tandem repeats containing motifs capable of assembling into very stable secondary structures that is likely responsible.


April 21, 2020  |  

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph “Candidatus Methylacidiphilum kamchatkense” strain Kam1 and comparison with its closest relatives.

The candidate genus “Methylacidiphilum” comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the “Ca. Methylacidiphilum” and the sister genus “Ca. Methylacidimicrobium” represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.Here we present the closed genome of “Ca. Methylacidiphilum kamchatkense” strain Kam1 and a comparison with the genomes of its two closest relatives “Ca. Methylacidiphilum fumariolicum” strain SolV and “Ca. Methylacidiphilum infernorum” strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of “Ca. Methylacidiphilum” is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between “Ca. M. kamchatkense” strain Kam1 and strains SolV and V4 is =95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.Comparing three closed genomes of “Ca. Methylacidiphilum” spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.


April 21, 2020  |  

A Pathovar of Xanthomonas oryzae Infecting Wild Grasses Provides Insight Into the Evolution of Pathogenicity in Rice Agroecosystems

Xanthomonas oryzae (Xo) are critical rice pathogens. Virulent lineages from Africa and Asia and less virulent strains from the US have been well characterized. X. campestris pv. leersiae (Xcl), first described in 1957, causes bacterial streak on the perennial grass, Leersia hexandra, and is a close relative of Xo. L. hexandra, a member of the Poaceae, is highly similar to rice phylogenetically, is globally ubiquitous around rice paddies, and is a reservoir of pathogenic Xo. We used long read, single molecule, real time (SMRT) genome sequences of five strains of Xcl from Burkina Faso, China, Mali and Uganda to determine the genetic relatedness of this organism with Xo. Novel Transcription Activator-Like Effectors (TALEs) were discovered in all five strains of Xcl. Predicted TALE target sequences were identified in the L. perrieri genome and compared to rice susceptibility gene homologs. Pathogenicity screening on L. hexandra and diverse rice cultivars confirmed that Xcl are able to colonize rice and produce weak but not progressive symptoms. Overall, based on average nucleotide identity, type III effector repertoires and disease phenotype, we propose to rename Xcl to X. oryzae pv. leersiae (Xol) and use this parallel system to improve understanding of the evolution of bacterial pathogenicity in rice agroecosystems.


April 21, 2020  |  

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.


April 21, 2020  |  

Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa.

Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C and D, three genes for LPS biosynthesis and had reduced antibiotic resistance genes when compared with clinical strains. Genes belonging to proteases, bacterial exporters and DNA stabilization were found to be under strong positive selection, thus facilitating pathogenicity and survival of the outliers. The outliers had the complete operon for the production of vibrioferrin, a siderophore present in plant growth promoting bacteria. The competence to acquire multidrug resistance and new virulence factors makes these strains a potential threat. However, we identified major regulatory hubs that can be used as drug targets against both the classical and outlier groups.


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  |  

High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system.

Coral reefs are a complex ecosystem consisting of coral animals and a vast array of associated symbionts including the dinoflagellate Symbiodinium, fungi, viruses and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their fundamental roles in fitness and survival of the host animal. The scleractinian coral Porites lutea is one of the dominant reef-builders in the Indo-West Pacific. Currently, very little is known about the composition and structure of bacterial communities across P. lutea reefs. The purpose of this study is twofold: to demonstrate the advantages of using PacBio circular consensus sequencing technology in microbial community studies and to investigate the diversity and structure of P. lutea-associated microbiome in the Indo-Pacific. This is the first metagenomic study of marine environmental samples that utilises the PacBio sequencing system to capture full-length 16S rRNA sequences. We observed geographically distinct coral-associated microbial profiles between samples from the Gulf of Thailand and Andaman Sea. Despite the geographical and environmental impacts on the coral-host interactions, we identified a conserved community of bacteria that were present consistently across diverse reef habitats. Finally, we demonstrated the superior performance of full-length 16S rRNA sequences in resolving taxonomic uncertainty of coral associates at the species level.


September 22, 2019  |  

Is there foul play in the leaf pocket? The metagenome of floating fern Azolla reveals endophytes that do not fix N2 but may denitrify.

Dinitrogen fixation by Nostoc azollae residing in specialized leaf pockets supports prolific growth of the floating fern Azolla filiculoides. To evaluate contributions by further microorganisms, the A. filiculoides microbiome and nitrogen metabolism in bacteria persistently associated with Azolla ferns were characterized. A metagenomic approach was taken complemented by detection of N2 O released and nitrogen isotope determinations of fern biomass. Ribosomal RNA genes in sequenced DNA of natural ferns, their enriched leaf pockets and water filtrate from the surrounding ditch established that bacteria of A. filiculoides differed entirely from surrounding water and revealed species of the order Rhizobiales. Analyses of seven cultivated Azolla species confirmed persistent association with Rhizobiales. Two distinct nearly full-length Rhizobiales genomes were identified in leaf-pocket-enriched samples from ditch grown A. filiculoides. Their annotation revealed genes for denitrification but not N2 -fixation. 15 N2 incorporation was active in ferns with N. azollae but not in ferns without. N2 O was not detectably released from surface-sterilized ferns with the Rhizobiales. N2 -fixing N. azollae, we conclude, dominated the microbiome of Azolla ferns. The persistent but less abundant heterotrophic Rhizobiales bacteria possibly contributed to lowering O2 levels in leaf pockets but did not release detectable amounts of the strong greenhouse gas N2 O.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.


September 22, 2019  |  

Identification of Burkholderia fungorum in the urine of an individual with spinal cord injury and augmentation cystoplasty using 16S sequencing: copathogen or innocent bystander?

People with neuropathic bladder (NB) secondary to spinal cord injury (SCI) are at risk for multiple genitourinary complications, the most frequent of which is urinary tract infection (UTI). Despite the high frequency with which UTI occurs, our understanding of the role of urinary microbes in health and disease is limited. In this paper, we present the first prospective case study integrating symptom reporting, urinalysis, urine cultivation, and 16S ribosomal ribonucleic acid (rRNA) sequencing of the urine microbiome.A 55-year-old male with NB secondary to SCI contributed 12 urine samples over an 8-month period during asymptomatic, symptomatic, and postantibiotic periods. All bacteria identified on culture were present on 16S rRNA sequencing, however, 16S rRNA sequencing revealed the presence of bacteria not isolated on culture. In particular, Burkholderia fungorum was present in three samples during both asymptomatic and symptomatic periods. White blood cells of =5-10/high power field and leukocyte esterase =2 on urinalysis was associated with the presence of symptoms.In this patient, there was a predominance of pathogenic bacteria and a lack of putative probiotic bacteria during both symptomatic and asymptomatic states. Urinalysis-defined inflammatory markers were present to a greater extent during symptomatic periods compared to the asymptomatic state, which may underscore a role for urinalysis or other inflammatory markers in differentiating asymptomatic bacteriuria from UTI in patients with NB. The finding of potentially pathogenic bacteria identified by sequencing but not cultivation, suggests a need for greater understanding of the relationships amongst bacterial species in the bacteriuric neuropathic bladder.


September 22, 2019  |  

MetaSort untangles metagenome assembly by reducing microbial community complexity.

Most current approaches to analyse metagenomic data rely on reference genomes. Novel microbial communities extend far beyond the coverage of reference databases and de novo metagenome assembly from complex microbial communities remains a great challenge. Here we present a novel experimental and bioinformatic framework, metaSort, for effective construction of bacterial genomes from metagenomic samples. MetaSort provides a sorted mini-metagenome approach based on flow cytometry and single-cell sequencing methodologies, and employs new computational algorithms to efficiently recover high-quality genomes from the sorted mini-metagenome by the complementary of the original metagenome. Through extensive evaluations, we demonstrated that metaSort has an excellent and unbiased performance on genome recovery and assembly. Furthermore, we applied metaSort to an unexplored microflora colonized on the surface of marine kelp and successfully recovered 75 high-quality genomes at one time. This approach will greatly improve access to microbial genomes from complex or novel communities.


September 22, 2019  |  

A response to Lindsey et al. “Wolbachia pipientis should not be split into multiple species: A response to Ramírez-Puebla et al.”.

In Ramírez-Puebla et al. [18] we compared 34 Wolbachia genomes and constructed phylogenetic trees using genomic data. In general, our results were congruent with previously reported phy- logenetic trees [5,9]. Our datasets were carefully selected, checked and analyzed avoiding horizontally transferred genes. In the case of the wAna genome we did not use the raw data, but the assem- bled genome [22] and 31 genes were used to compare in a dataset of conserved proteins. To confirm our conclusions a new phyloge- nomic analysis was performed excluding the wAna strain in the dataset (Fig. 1). The same topology was obtained, therefore indi- cating that the results were not affected by the presence of this particular strain.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.