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September 22, 2019

Characterization of the SN35N strain-specific exopolysaccharide encoded in the whole circular genome of a plant-derived Lactobacillus plantarum.

Lactobacillus plantarum SN35N, which has been previously isolated from pear, secretes exopolysaccharide (EPS). The aim of the present study is to characterize the EPS chemically and to find the EPS-biosynthesizing gene cluster. The present study demonstrates that the strain produces an acidic EPS carrying phosphate residue, which is composed of glucose, galactose, and mannose at a molecular ratio of 15.0?:?5.7?:?1.0. We also show that acidic EPS strongly inhibits the catalytic activity of hyaluronidase (EC 3.2.1.35), promoting an inflammatory reaction. In the present study, we also determined the complete genome sequence of the SN35N strain, demonstrating that the genome is a circular DNA with 3267626?bp, and the number of predicted coding genes is 3146, with a GC content of 44.51%. In addition, the strain harbors four plasmids, designated pSN35N-1, -2, -3, and -4. Although four EPS-biosynthesizing genes, designated lpe1, lpe2, lpe3, and lpe4, are present in the SN35N chromosomal DNA, another EPS gene cluster, lpe5, is located in the pSN35N-3 plasmid, composed of 35425?bp. EPS low-producing mutants, which were obtained by treating SN35N cells with novobiocin, lost the lpe5 gene cluster in the plasmid-curing experiment, suggesting that the gene cluster for the biosynthesis of acidic EPS is present in the plasmid. The present study shows the chemical characterization of the acidic EPS and its inhibitory effect to the hyaluronidase.

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September 22, 2019

Comparative genomic analyses reveal the features for adaptation to nematodes in fungi.

Nematophagous (NP) fungi are ecologically important components of the soil microbiome in natural ecosystems. Esteya vermicola (Ev) has been reported as a NP fungus with a poorly understood evolutionary history and mechanism of adaptation to parasitism. Furthermore, NP fungal genomic basis of lifestyle was still unclear. We sequenced and annotated the Ev genome (34.2 Mbp) and integrated genetic makeup and evolution of pathogenic genes to investigate NP fungi. The results revealed that NP fungi had some abundant pathogenic genes corresponding to their niche. A number of gene families involved in pathogenicity were expanded, and some pathogenic orthologous genes underwent positive selection. NP fungi with diverse morphological features exhibit similarities of evolutionary convergence in attacking nematodes, but their genetic makeup and microscopic mechanism are different. Endoparasitic NP fungi showed similarity in large number of transporters and secondary metabolite coding genes. Noteworthy, expanded families of transporters and endo-beta-glucanase implied great genetic potential of Ev in quickly perturbing nematode metabolism and parasitic behavior. These results facilitate our understanding of NP fungal genomic features for adaptation to nematodes and lay a solid theoretical foundation for further research and application.© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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September 22, 2019

An ancient integration in a plant NLR is maintained as a trans-species polymorphism

Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens. Nucleotide-binding leucine-rich repeat (NLR) proteins are one class of cytoplasmic immune receptors whose genes commonly show signatures of adaptive evolution. While it is known that balancing selection contributes to maintaining high intraspecific allelic diversity, the evolutionary mechanism that influences the transmission of alleles during speciation remains unclear. The barley Mla locus has over 30 described alleles conferring isolate-specific resistance to barley powdery mildew and contains three NLR families (RGH1, RGH2, and RGH3). We discovered (using sequence capture and RNAseq) the presence of a novel integrated Exo70 domain in RGH2 in the Mla3 haplotype. Allelic variation across barley accessions includes presence/absence of the integrated domain in RGH2. Expanding our search to several Poaceae species, we found shared interspecific conservation in the RGH2-Exo70 integration. We hypothesise that balancing selection has maintained allelic variation at Mla as a trans-species polymorphism over 24 My, thus contributing to and preserving interspecific allelic diversity during speciation.

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September 22, 2019

Screening and genomic characterization of filamentous hemagglutinin-deficient Bordetella pertussis.

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Characterization of plasmids harboring blaCTX-M and blaCMY genes in E. coli from French broilers.

Resistance to extended-spectrum cephalosporins (ESC) is a global health issue. The aim of this study was to analyze and compare plasmids coding for resistance to ESC isolated from 16 avian commensal and 17 avian pathogenic Escherichia coli (APEC) strains obtained respectively at slaughterhouse or from diseased broilers in 2010-2012. Plasmid DNA was used to transform E. coli DH5alpha, and the resistances of the transformants were determined. The sequences of the ESC-resistance plasmids prepared from transformants were obtained by Illumina (33 plasmids) or PacBio (1 plasmid). Results showed that 29 of these plasmids contained the blaCTX-M-1 gene and belonged to the IncI1/ST3 type, with 27 and 20 of them carrying the sul2 or tet(A) genes respectively. Despite their diverse origins, several plasmids showed very high percentages of identity. None of the blaCTX-M-1-containing plasmid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the blaCMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The blaCMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a blaCTX-M-1 IncI1/ST3 plasmid. Importantly APEC virulence genes (sitA-D, iucA-D, iutA, hlyF, ompT, etsA-C, iss, iroB-E, iroN, cvaA-C and cvi) were detected on the blaCMY-2 plasmid. In conclusion, our results show the dominance and high similarity of blaCTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a blaCMY-2 plasmid.

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September 22, 2019

The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology.

We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest,Trichoplusia ni, assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. Chemoreception and detoxification gene families revealT. ni-specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Transcriptome and small RNA data from thorax, ovary, testis, and the germline-derived Hi5 cell line show distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding small RNA pathway proteins. Nearly all of the W chromosome is devoted to piRNA production, andT. nisiRNAs are not 2´-O-methylated. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. TheT. nigenome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo.© 2018, Fu et al.

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September 22, 2019

Microbiome and infectivity studies reveal complex polyspecies tree disease in Acute Oak Decline.

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch’s postulates to address the role of microbial communities in disease.

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September 22, 2019

The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a “Y. ruckeri invasin-like molecule”, (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen.

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel Limnoperna fortunei.

For more than 25 years, the golden mussel Limnoperna fortunei has aggressively invaded South American freshwaters, having travelled more than 5,000 km upstream across five countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it.We assembled the 1.6 Gb genome into 20548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions: We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina pair-end, mate pair, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.© The Authors 2017. Published by Oxford University Press.

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September 22, 2019

Genome sequences of Chlorella sorokiniana UTEX 1602 and Micractinium conductrix SAG 241.80: implications to maltose excretion by a green alga.

Green algae represent a key segment of the global species capable of photoautotrophic-driven biological carbon fixation. Algae partition fixed-carbon into chemical compounds required for biomass, while diverting excess carbon into internal storage compounds such as starch and lipids or, in certain cases, into targeted extracellular compounds. Two green algae were selected to probe for critical components associated with sugar production and release in a model alga. Chlorella sorokiniana UTEX 1602 – which does not release significant quantities of sugars to the extracellular space – was selected as a control to compare with the maltose-releasing Micractinium conductrix SAG 241.80 – which was originally isolated from an endosymbiotic association with the ciliate Paramecium bursaria. Both strains were subjected to three sequencing approaches to assemble their genomes and annotate their genes. This analysis was further complemented with transcriptional studies during maltose release by M. conductrix SAG 241.80 versus conditions where sugar release is minimal. The annotation revealed that both strains contain homologs for the key components of a putative pathway leading to cytosolic maltose accumulation, while transcriptional studies found few changes in mRNA levels for the genes associated with these established intracellular sugar pathways. A further analysis of potential sugar transporters found multiple homologs for SWEETs and tonoplast sugar transporters. The analysis of transcriptional differences revealed a lesser and more measured global response for M. conductrix SAG 241.80 versus C. sorokiniana UTEX 1602 during conditions resulting in sugar release, providing a catalog of genes that might play a role in extracellular sugar transport.© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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September 22, 2019

Plasmid-encoded transferable mecB-mediated methicillin resistance in Staphylococcus aureus.

During cefoxitin-based nasal screening, phenotypically categorized methicillin-resistant Staphylococcus aureus (MRSA) was isolated and tested negative for the presence of the mecA and mecC genes as well as for the SCCmec-orfX junction region. The isolate was found to carry a mecB gene previously described for Macrococcus caseolyticus but not for staphylococcal species. The gene is flanked by ß-lactam regulatory genes similar to mecR, mecI, and blaZ and is part of an 84.6-kb multidrug-resistance plasmid that harbors genes encoding additional resistances to aminoglycosides (aacA-aphD, aphA, and aadK) as well as macrolides (ermB) and tetracyclines (tetS). This further plasmidborne ß-lactam resistance mechanism harbors the putative risk of acceleration or reacceleration of MRSA spread, resulting in broad ineffectiveness of ß-lactams as a main therapeutic application against staphylococcal infections.

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September 22, 2019

Reference quality genome assemblies of three Parastagonospora nodorum isolates differing in virulence on wheat.

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch in wheat, has emerged as a model necrotrophic fungal organism for the study of host-microbe interactions. To date, three necrotrophic effectors have been identified and characterized from this pathogen, including SnToxA, SnTox1, and SnTox3. Necrotrophic effector identification was greatly aided by the development of a draft genome of Australian isolate SN15 via Sanger sequencing, yet it remained largely fragmented. This research presents the development of nearly finished genomes of P. nodorum isolates Sn4, Sn2000, and Sn79-1087 using long-read sequencing technology. RNAseq analysis of isolate Sn4, consisting of eight time points covering various developmental and infection stages, mediated the annotation of 13,379 genes. Analysis of these genomes revealed large-scale polymorphism between the three isolates, including the complete absence of contig 23 from isolate Sn79-1087, and a region of genome expansion on contig 10 in isolates Sn4 and Sn2000. Additionally, these genomes exhibit the hallmark characteristics of a “two-speed” genome, being partitioned into two distinct GC-equilibrated and AT-rich compartments. Interestingly, isolate Sn79-1087 contains a lower proportion of AT-rich segments, indicating a potential lack of evolutionary hotspots. These newly sequenced genomes, consisting of telomere-to-telomere assemblies of nearly all 23 P. nodorum chromosomes, provide a robust foundation for the further examination of effector biology and genome evolution. Copyright © 2018 Richards et al.

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September 22, 2019

Blood CXCR3+CD4 T cells are enriched in inducible replication competent HIV in aviremic antiretroviral therapy-treated individuals.

We recently demonstrated that lymph nodes (LNs) PD-1+/T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1+CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1+CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

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September 22, 2019

Pseudomonas orientalis F9: A potent antagonist against phytopathogens with phytotoxic effect in the apple flower.

In light of public concerns over the use of pesticides and antibiotics in plant protection and the subsequent selection for spread of resistant bacteria in the environment, it is inevitable to broaden our knowledge about viable alternatives, such as natural antagonists and their mode of action. The genus Pseudomonas is known for its metabolic versatility and genetic plasticity, encompassing pathogens as well as antagonists. We characterized strain Pseudomonas orientalis F9, an isolate from apple flowers in a Swiss orchard, and determined its antagonistic activity against several phytopathogenic bacteria, in particular Erwinia amylovora, the causal agent of fire blight. P. orientalis F9 displayed antagonistic activity against a broad suite of phytopathogenic bacteria in the in vitro tests. The promising results from this analysis led to an ex vivo assay with E. amylovora CFBP1430Rif and P. orientalis F9 infected detached apple flowers. F9 diminished the fire blight pathogen in the flowers but also revealed phytotoxic traits. The experimental results were discussed in light of the complete genome sequence of F9, which revealed the strain to carry phenazine genes. Phenazines are known to contribute to antagonistic activity of bacterial strains against soil pathogens. When tested in the cress assay with Pythium ultimum as pathogen, F9 showed results comparable to the known antagonist P. protegens CHA0.

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September 22, 2019

Analyzing AbrB-knockout effects through genome and transcriptome sequencing of Bacillus licheniformis DW2.

As an industrial bacterium, Bacillus licheniformis DW2 produces bacitracin which is an important antibiotic for many pathogenic microorganisms. Our previous study showed AbrB-knockout could significantly increase the production of bacitracin. Accordingly, it was meaningful to understand its genome features, expression differences between wild and AbrB-knockout (?AbrB) strains, and the regulation of bacitracin biosynthesis. Here, we sequenced, de novo assembled and annotated its genome, and also sequenced the transcriptomes in three growth phases. The genome of DW2 contained a DNA molecule of 4,468,952 bp with 45.93% GC content and 4,717 protein coding genes. The transcriptome reads were mapped to the assembled genome, and obtained 4,102~4,536 expressed genes from different samples. We investigated transcription changes in B. licheniformis DW2 and showed that ?AbrB caused hundreds of genes up-regulation and down-regulation in different growth phases. We identified a complete bacitracin synthetase gene cluster, including the location and length of bacABC, bcrABC, and bacT, as well as their arrangement. The gene cluster bcrABC were significantly up-regulated in ?AbrB strain, which supported the hypothesis in previous study of bcrABC transporting bacitracin out of the cell to avoid self-intoxication, and was consistent with the previous experimental result that ?AbrB could yield more bacitracin. This study provided a high quality reference genome for B. licheniformis DW2, and the transcriptome data depicted global alterations across two strains and three phases offered an understanding of AbrB regulation and bacitracin biosynthesis through gene expression.

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