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September 22, 2019

Comparison of highly and weakly virulent Dickeya solani strains, with a view on the pangenome and panregulon of this species.

Bacteria belonging to the genera Dickeya and Pectobacterium are responsible for significant economic losses in a wide variety of crops and ornamentals. During last years, increasing losses in potato production have been attributed to the appearance of Dickeya solani. The D. solani strains investigated so far share genetic homogeneity, although different virulence levels were observed among strains of various origins. The purpose of this study was to investigate the genetic traits possibly related to the diverse virulence levels by means of comparative genomics. First, we developed a new genome assembly pipeline which allowed us to complete the D. solani genomes. Four de novo sequenced and ten publicly available genomes were used to identify the structure of the D. solani pangenome, in which 74.8 and 25.2% of genes were grouped into the core and dispensable genome, respectively. For D. solani panregulon analysis, we performed a binding site prediction for four transcription factors, namely CRP, KdgR, PecS and Fur, to detect the regulons of these virulence regulators. Most of the D. solani potential virulence factors were predicted to belong to the accessory regulons of CRP, KdgR, and PecS. Thus, some differences in gene expression could exist between D. solani strains. The comparison between a highly and a low virulent strain, IFB0099 and IFB0223, respectively, disclosed only small differences between their genomes but significant differences in the production of virulence factors like pectinases, cellulases and proteases, and in their mobility. The D. solani strains also diverge in the number and size of prophages present in their genomes. Another relevant difference is the disruption of the adhesin gene fhaB2 in the highly virulent strain. Strain IFB0223, which has a complete adhesin gene, is less mobile and less aggressive than IFB0099. This suggests that in this case, mobility rather than adherence is needed in order to trigger disease symptoms. This study highlights the utility of comparative genomics in predicting D. solani traits involved in the aggressiveness of this emerging plant pathogen.

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September 22, 2019

Genomic analysis for heavy metal resistance in S. maltophilia

Stenotrophomonas maltophilia is highly resistant to heavy metals, but the genetic knowledge of metal resistance in S. maltophilia is poorly understood. In this study, the genome of S. maltophilia Pho isolated from the contaminated soil near a metalwork factory was sequenced using PacBio RS II. Its genome is composed of a single chromosome with a GC content of 66.4% and 4434 protein-encoding genes. Comparative analysis revealed high syntney between S. maltophilia Pho and the model strain, S. maltophilia K279a. Then, the type and number of mechanisms of heavy metal uptake were analyzed firstly. Results showed that 7 unspecific ion transporter genes and 13 specific ion transporter genes, most of which were involved in iron transport. But the sulfate permeases belonging to the family of SulT/CysP that can uptake chromate and the high affinity ZnuABC/SitABCD were absent. Secondly, the putative genes controlling metal efflux were analyzed. Results showed that this bacterium encoded 5 CDFs, 1 copper exporting ATPase and 4 RND systems, including 2 CzcABC efflux pumps. Moreover, the putative metal transformation genes including arsenate and mercury detoxification genes were also identified. This study may provide useful information on the metal resistance mechanisms of S. maltophilia.

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September 22, 2019

Variant O89 O-antigen of E. coli is associated with group 1 capsule loci and multidrug resistance.

Bacterial surface polysaccharides play significant roles in fitness and virulence. In Gram-negative bacteria such as Escherichia coli, major surface polysaccharides are lipopolysaccharide (LPS) and capsule, representing O- and K-antigens, respectively. There are multiple combinations of O:K types, many of which are well-characterized and can be related to ecotype or pathotype. In this investigation, we have identified a novel O:K permutation resulting through a process of major genome reorganization in a clade of E. coli. A multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing strain – E. coli 26561 – represented a prototype of strains combining a locus variant of O89 and group 1 capsular polysaccharide. Specifically, the variant O89 locus in this strain was truncated at gnd, flanked by insertion sequences and located between nfsB and ybdK and we apply the term O89m for this variant. The prototype lacked colanic acid and O-antigen loci between yegH and hisI with this tandem polysaccharide locus being replaced with a group 1 capsule (G1C) which, rather than being a recognized E. coli capsule type, this locus matched to Klebsiella K10 capsule type. A genomic survey identified more than 200 E. coli strains which possessed the O89m locus variant with one of a variety of G1C types. Isolates from our collection with the combination of O89m and G1C all displayed a mucoid phenotype and E. coli 26561 was unusual in exhibiting a mucoviscous phenotype more recognized as a characteristic among Klebsiella strains. Despite the locus truncation and novel location, all O89m:G1C strains examined showed a ladder pattern typifying smooth LPS and also showed high molecular weight, alcian blue-staining polysaccharide in cellular and/or extra-cellular fractions. Expression of both O-antigen and capsule biosynthesis loci were confirmed in prototype strain 26561 through quantitative proteome analysis. Further in silico exploration of more than 200 E. coli strains possessing the O89m:G1C combination identified a very high prevalence of multidrug resistance (MDR) – 85% possessed resistance to three or more antibiotic classes and a high proportion (58%) of these carried ESBL and/or carbapenemase. The increasing isolation of O89m:G1C isolates from extra-intestinal infection sites suggests that these represents an emergent clade of invasive, MDR E. coli.

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September 22, 2019

Genomics of Corynebacterium striatum, an emerging multidrug-resistant pathogen of immunocompromised patients.

Corynebacterium striatum is an emerging multidrug-resistant (MDR) pathogen of immunocompromised and chronically ill patients. The objective of these studies was to provide a detailed genomic analysis of disease-causing C. striatum and determine the genomic drivers of resistance and resistance-gene transmission.A multi-institutional and prospective pathogen genomics programme flagged seven MDR C. striatum infections occurring close in time, and specifically in immunocompromised patients with underlying respiratory diseases. Whole genome sequencing was used to identify clonal relationships among strains, genetic causes of antimicrobial resistance, and their mobilization capacity. Matrix-assisted linear desorption/ionization-time-of-flight analyses of sequenced isolates provided curated content to improve rapid clinical identification in subsequent cases.Epidemiological and genomic analyses identified a related cluster of three out of seven C. striatum among lung transplant patients who had common procedures and exposures at an outlying institution. Genomic analyses further elucidated drivers of the MDR phenotypes, including resistance genes mobilized by IS3504 and ISCg9a-like insertion sequences. Seven mobilizable resistance genes were localized to a common chromosomal region bounded by unpaired insertion sequences, suggesting that a single recombination event could spread resistance to aminoglycosides, macrolides, lincosamides and tetracyclines to naive strains.In-depth genomic studies of MDR C. striatum reveal its capacity for clonal spread within and across healthcare institutions and identify novel vectors that can mobilize multiple forms of drug resistance, further complicating efforts to treat infections in immunocompromised populations. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

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September 22, 2019

Involvement of Burkholderiaceae and sulfurous volatiles in disease-suppressive soils.

Disease-suppressive soils are ecosystems in which plants suffer less from root infections due to the activities of specific microbial consortia. The characteristics of soils suppressive to specific fungal root pathogens are comparable to those of adaptive immunity in animals, as reported by Raaijmakers and Mazzola (Science 352:1392-3, 2016), but the mechanisms and microbial species involved in the soil suppressiveness are largely unknown. Previous taxonomic and metatranscriptome analyses of a soil suppressive to the fungal root pathogen Rhizoctonia solani revealed that members of the Burkholderiaceae family were more abundant and more active in suppressive than in non-suppressive soils. Here, isolation, phylogeny, and soil bioassays revealed a significant disease-suppressive activity for representative isolates of Burkholderia pyrrocinia, Paraburkholderia caledonica, P. graminis, P. hospita, and P. terricola. In vitro antifungal activity was only observed for P. graminis. Comparative genomics and metabolite profiling further showed that the antifungal activity of P. graminis PHS1 was associated with the production of sulfurous volatile compounds encoded by genes not found in the other four genera. Site-directed mutagenesis of two of these genes, encoding a dimethyl sulfoxide reductase and a cysteine desulfurase, resulted in a loss of antifungal activity both in vitro and in situ. These results indicate that specific members of the Burkholderiaceae family contribute to soil suppressiveness via the production of sulfurous volatile compounds.

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September 22, 2019

Genome annotation and comparative genomic analysis of Bacillus subtilis MJ01, a new bio-degradation strain isolated from oil-contaminated soil.

One of the main challenges in elimination of oil contamination from polluted environments is improvement of biodegradation by highly efficient microorganisms. Bacillus subtilis MJ01 has been evaluated as a new resource for producing biosurfactant compounds. This bacterium, which produces surfactin, is able to enhance bio-accessibility to oil hydrocarbons in contaminated soils. The genome of B. subtilis MJ01 was sequenced and assembled by PacBio RS sequencing technology. One big contig with a length of 4,108,293 bp without any gap was assembled. Genome annotation and prediction of gene showed that MJ01 genome is very similar to B. subtilis spizizenii TU-B-10 (95% similarity). The comparison and analysis of orthologous genes carried out between B. subtilis MJ01, reference strain B. subtilis subsp. subtilis str. 168, and close relative spizizenii TU-B-10 by microscope platform and various bioinformatics tools. More than 88% of 4269 predicted coding sequences in MJ01 had at least one similar sequence in genome of reference strain and spizizenii TU-B-10. Despite this high similarity, some differences were detected among encoding sequences of non-ribosome protein and bacteriocins in MJ01 and spizizenii TU-B-10. MJ01 has unique nucleotide sequences and a novel predicted lasso-peptide bacteriocin; it also has not any similar nucleotide sequence in non-redundant nucleotide data base.

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September 22, 2019

Prevalence and genomic structure of bacteriophage phi3 in human derived livestock-associated MRSA from 2000 to 2015.

Whereas the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex 398 (CC398) in animal husbandry and its transmission to humans are well documented, less is known about factors driving the epidemic spread of this zoonotic lineage within the human population. One factor could be the bacteriophage phi3, which is rarely detected in S. aureus isolates from animals but commonly found among isolates from humans, including those of the human-adapted methicillin-susceptible S. aureus (MSSA) CC398 clade. The proportion of phi3-carrying MRSA spa-CC011 isolates, which constitute presumptively LA-MRSA within the multilocus sequence type (MLST) clonal complex 398, was systematically assessed for a period of 16 years to investigate the role of phi3 in the adaptation process of LA-MRSA to the human host. For this purpose, 632 MRSA spa-CC011 isolates from patients of a university hospital located in a pig farming-dense area in Germany were analyzed. Livestock-associated acquisition of MRSA spa-CC011 was previously reported as having increased from 1.8% in 2000 to 29.4% in 2014 in MRSA-positive patients admitted to this hospital. However, in this study, the proportion of phi3-carrying isolates rose only from 1.1% (2000 to 2006) to 3.9% (2007 to 2015). Characterization of the phi3 genomes revealed 12 different phage types ranging in size from 40,712 kb up to 44,003 kb, with four hitherto unknown integration sites (genes or intergenic regions) and several modified bacterial attachment (attB) sites. In contrast to the MSSA CC398 clade, phi3 acquisition seems to be no major driver for the readaptation of MRSA spa-CC011 to the human host. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

The chromosome-level genome assemblies of two rattans (Calamus simplicifolius and Daemonorops jenkinsiana).

Calamus simplicifolius and Daemonorops jenkinsiana are two representative rattans, the most significant material sources for the rattan industry. However, the lack of reference genome sequences is a major obstacle for basic and applied biology on rattan.We produced two chromosome-level genome assemblies of C. simplicifolius and D. jenkinsiana using Illumina, Pacific Biosciences, and Hi-C sequencing data. A total of ~730 Gb and ~682 Gb of raw data covered the predicted genome lengths (~1.98 Gb of C. simplicifolius and ~1.61 Gb of D. jenkinsiana) to ~372 × and ~426 × read depths, respectively. The two de novo genome assemblies, ~1.94 Gb and ~1.58 Gb, were generated with scaffold N50s of ~160 Mb and ~119 Mb in C. simplicifolius and D. jenkinsiana, respectively. The C. simplicifolius and D. jenkinsiana genomes were predicted to harbor ?51,235 and ?53,342 intact protein-coding gene models, respectively. Benchmarking Universal Single-Copy Orthologs evaluation demonstrated that genome completeness reached 96.4% and 91.3% in the C. simplicifolius and D. jenkinsiana genomes, respectively. Genome evolution showed that four Arecaceae plants clustered together, and the divergence time between the two rattans was ~19.3 million years ago. Additionally, we identified 193 and 172 genes involved in the lignin biosynthesis pathway in the C. simplicifolius and D. jenkinsiana genomes, respectively.We present the first de novo assemblies of two rattan genomes (C. simplicifolius and D. jenkinsiana). These data will not only provide a fundamental resource for functional genomics, particularly in promoting germplasm utilization for breeding, but also serve as reference genomes for comparative studies between and among different species.

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September 22, 2019

Draft genome assembly of the invasive cane toad, Rhinella marina.

The cane toad (Rhinella marina formerly Bufo marinus) is a species native to Central and South America that has spread across many regions of the globe. Cane toads are known for their rapid adaptation and deleterious impacts on native fauna in invaded regions. However, despite an iconic status, there are major gaps in our understanding of cane toad genetics. The availability of a genome would help to close these gaps and accelerate cane toad research.We report a draft genome assembly for R. marina, the first of its kind for the Bufonidae family. We used a combination of long-read Pacific Biosciences RS II and short-read Illumina HiSeq X sequencing to generate 359.5 Gb of raw sequence data. The final hybrid assembly of 31,392 scaffolds was 2.55 Gb in length with a scaffold N50 of 168 kb. BUSCO analysis revealed that the assembly included full length or partial fragments of 90.6% of tetrapod universal single-copy orthologs (n = 3950), illustrating that the gene-containing regions have been well assembled. Annotation predicted 25,846 protein coding genes with similarity to known proteins in Swiss-Prot. Repeat sequences were estimated to account for 63.9% of the assembly.The R. marina draft genome assembly will be an invaluable resource that can be used to further probe the biology of this invasive species. Future analysis of the genome will provide insights into cane toad evolution and enrich our understanding of their interplay with the ecosystem at large.

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September 22, 2019

Draft genome of Glyptosternon maculatum, an endemic fish from Tibet Plateau.

Mechanisms for high-altitude adaption have attracted widespread interest among evolutionary biologists. Several genome-wide studies have been carried out for endemic vertebrates in Tibet, including mammals, birds, and amphibians. However, little information is available about the adaptive evolution of highland fishes. Glyptosternon maculatum (Regan 1905), also known as Regan or barkley and endemic to the Tibetan Plateau, belongs to the Sisoridae family, order Siluriformes (catfishes). This species lives at an elevation ranging from roughly 2,800 m to 4,200 m. Hence, a high-quality reference genome of G. maculatum provides an opportunity to investigate high-altitude adaption mechanisms of fishes.To obtain a high-quality reference genome sequence of G. maculatum, we combined Pacific Bioscience single-molecule real-time sequencing, Illumina paired-end sequencing, 10X Genomics linked-reads, and BioNano optical map techniques. In total, 603.99 Gb sequencing data were generated. The assembled genome was about 662.34 Mb with scaffold and contig N50 sizes of 20.90 Mb and 993.67 kb, respectively, which captured 83% complete and 3.9% partial vertebrate Benchmarking Universal Single-Copy Orthologs. Repetitive elements account for 35.88% of the genome, and ?22,066 protein-coding genes were predicted from the genome, of which 91.7% have been functionally annotated.We present the first comprehensive de novo genome of G. maculatum. This genetic resource is fundamental for investigating the origin of G. maculatum and will improve our understanding of high-altitude adaption of fishes. The assembled genome can also be used as reference for future population genetic studies of G. maculatum.

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September 22, 2019

The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri.

Members of the genus Naegleria are free-living eukaryotes with the capability to transform from the amoeboid form into resting cysts or moving flagellates in response to environmental conditions. More than 40 species have been characterized, but only Naegleria fowleri (N. fowleri) is known as a human pathogen causing primary amoebic meningoencephalitis (PAM), a fast progressing and mostly fatal disease of the central nervous system. Several studies report an involvement of phospholipases and other molecular factors, but the mechanisms involved in pathogenesis are still poorly understood. To gain a better understanding of the relationships within the genus of Naegleria and to investigate pathogenicity factors of N. fowleri, we characterized the genome of its closest non-pathogenic relative N. lovaniensis.To gain insights into the taxonomy of Naegleria, we sequenced the genome of N. lovaniensis using long read sequencing technology. The assembly of the data resulted in a 30 Mb genome including the circular mitochondrial sequence. Unravelling the phylogenetic relationship using OrthoMCL protein clustering and maximum likelihood methods confirms the close relationship of N. lovaniensis and N. fowleri. To achieve an overview of the diversity of Naegleria proteins and to assess characteristics of the human pathogen N. fowleri, OrthoMCL protein clustering including data of N. fowleri, N. lovaniensis and N. gruberi was performed. GO enrichment analysis shows an association of N. fowleri specific proteins to the GO terms “Membrane” and “Protein Secretion.”In this study, we characterize the hitherto unknown genome of N. lovaniensis. With the description of the 30 Mb genome, a further piece is added to reveal the complex taxonomic relationship of Naegleria. Further, the whole genome sequencing data confirms the hypothesis of the close relationship between N. fowleri and N. lovaniensis. Therefore, the genome of N. lovaniensis provides the basis for further comparative approaches on the molecular and genomic level to unravel pathogenicity factors of its closest human pathogenic relative N. fowleri and possible treatment options for the rare but mostly fatal primary meningoencephalitis.

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September 22, 2019

Genome analyses of the microalga Picochlorum provide insights into the evolution of thermotolerance in the green lineage.

While the molecular events involved in cell responses to heat stress have been extensively studied, our understanding of the genetic basis of basal thermotolerance, and particularly its evolution within the green lineage, remains limited. Here, we present the 13.3-Mb haploid genome and transcriptomes of a halotolerant and thermotolerant unicellular green alga, Picochlorum costavermella (Trebouxiophyceae) to investigate the evolution of the genomic basis of thermotolerance. Differential gene expression at high and standard temperatures revealed that more of the gene families containing up-regulated genes at high temperature were recently evolved, and less originated at the ancestor of green plants. Inversely, there was an excess of ancient gene families containing transcriptionally repressed genes. Interestingly, there is a striking overlap between the thermotolerance and halotolerance transcriptional rewiring, as more than one-third of the gene families up-regulated at 35?°C were also up-regulated under variable salt concentrations in Picochlorum SE3. Moreover, phylogenetic analysis of the 9,304 protein coding genes revealed 26 genes of horizontally transferred origin in P. costavermella, of which five were differentially expressed at higher temperature. Altogether, these results provide new insights about how the genomic basis of adaptation to halo- and thermotolerance evolved in the green lineage.

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September 22, 2019

Genomic insights into host adaptation between the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) and the barley stripe rust pathogen (Puccinia striiformis f. sp. hordei).

Plant fungal pathogens can rapidly evolve and adapt to new environmental conditions in response to sudden changes of host populations in agro-ecosystems. However, the genomic basis of their host adaptation, especially at the forma specialis level, remains unclear.We sequenced two isolates each representing Puccinia striiformis f. sp. tritici (Pst) and P. striiformis f. sp. hordei (Psh), different formae speciales of the stripe rust fungus P. striiformis highly adapted to wheat and barley, respectively. The divergence of Pst and Psh, estimated to start 8.12 million years ago, has been driven by high nucleotide mutation rates. The high genomic variation within dikaryotic urediniospores of P. striiformis has provided raw genetic materials for genome evolution. No specific gene families have enriched in either isolate, but extensive gene loss events have occurred in both Pst and Psh after the divergence from their most recent common ancestor. A large number of isolate-specific genes were identified, with unique genomic features compared to the conserved genes, including 1) significantly shorter in length; 2) significantly less expressed; 3) significantly closer to transposable elements; and 4) redundant in pathways. The presence of specific genes in one isolate (or forma specialis) was resulted from the loss of the homologues in the other isolate (or forma specialis) by the replacements of transposable elements or losses of genomic fragments. In addition, different patterns and numbers of telomeric repeats were observed between the isolates.Host adaptation of P. striiformis at the forma specialis level is a complex pathogenic trait, involving not only virulence-related genes but also other genes. Gene loss, which might be adaptive and driven by transposable element activities, provides genomic basis for host adaptation of different formae speciales of P. striiformis.

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September 22, 2019

The transducer-like protein Tlp12 of Campylobacter jejuni is involved in glutamate and pyruvate chemotaxis.

Campylobacter jejuni is one of the most common bacterial causes of food-borne enteritis worldwide. Chemotaxis in C. jejuni is known to be critical for the successful colonization of the host and key for the adaptation of the microbial species to different host environments. In C. jejuni, chemotaxis is regulated by a complex interplay of 13 or even more different chemoreceptors, also known as transducer-like proteins (Tlps). Recently, a novel chemoreceptor gene, tlp12, was described and found to be present in 29.5% of the investigated C. jejuni strains.In this study, we present a functional analysis of Tlp12 with the aid of a tlp12 knockout mutant of the C. jejuni strain A17. Substrate specificity was investigated by capillary chemotaxis assays and revealed that Tlp12 plays an important role in chemotaxis towards glutamate and pyruvate. Moreover, the ?tlp12 mutant shows increased swarming motility in soft agar assays, an enhanced invasion rate into Caco-2 cells and an increased autoagglutination rate. The growth rate was slightly reduced in the ?tlp12 mutant. The identified phenotypes were in partial restored by complementation with the wild type gene. Tlp12-harboring C. jejuni strains display a strong association with chicken, whose excreta are known to contain high glutamate levels.TLP12 is a chemoreceptor for glutamate and pyruvate recognition. Deletion of tlp12 has an influence on distinct physiological features, such as growth rate, swarming motility, autoagglutination and invasiveness.

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September 22, 2019

Genes significantly associated with lineage II food isolates of Listeria monocytogenes.

Listeria monocytogenes is a widespread foodborne pathogen that can cause listeriosis, a potentially fatal infection. L. monocytogenes is subdivided into four phylogenetic lineages, with the highest incidence of listeriosis occurring within lineage I followed by lineage II. Strains of L. monocytogenes differ in their phenotypic characteristics, including virulence. However, the genetic bases for these observed differences are not well understood, and current efforts to monitor L. monocytogenes in food consider all strains to be equally virulent. We use a comparative genomics approach to identify genes and single nucleotide polymorphisms (SNPs) in 174 clinical and food isolates of L. monocytogenes that potentially contribute to virulence or the capacity to adapt to food environments.No SNPs are significantly associated with food or clinical isolates. No genes are significantly associated with food or clinical isolates from lineage I, but eight genes consisting of multiple homologues are associated with lineage II food isolates. These include three genes which encode hypothetical proteins, the cadmium resistance genes cadA and cadC, the multi-drug resistance gene ebrB, a quaternary ammonium compound resistance gene qac, and a regulatory gene. All eight genes are plasmid-borne, and most closed L. monocytogenes plasmids carry at least five of the genes (24/27). In addition, plasmids are more frequently associated with lineage II food isolates than with lineage II clinical isolates.We identify eight genes that are significantly associated with food isolates in lineage II. Interestingly, the eight genes are virtually absent in lineage II outbreak isolates, are composed of homologues which show a nonrandom distribution among lineage I serotypes, and the sequences are highly conserved across 27 closed Listeria plasmids. The functions of these genes should be explored further and will contribute to our understanding of how L. monocytogenes adapts to the host and food environments. Moreover, these genes may also be useful as markers for risk assessment models of either pathogenicity or the ability to proliferate in food and the food processing environment.

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