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July 7, 2019

Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models.

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July 7, 2019

Novel urease-negative Helicobacter sp. ‘H. enhydrae sp. nov.’ isolated from inflamed gastric tissue of southern sea otters.

A total of 31 sea otters Enhydra lutris nereis found dead or moribund (and then euthanized) were necropsied in California, USA. Stomach biopsies were collected and transected with equal portions frozen or placed in formalin and analyzed histologically and screened for Helicobacter spp. in gastric tissue. Helicobacter spp. were isolated from 9 sea otters (29%); 58% (18 of 31) animals were positive for helicobacter by PCR. The Helicobacter sp. was catalase- and oxidase-positive and urease-negative. By electron microscopy, the Helicobacter sp. had lateral and polar sheathed flagella and had a slightly curved rod morphology. 16S and 23S rRNA sequence analyses of all ‘H. enhydrae’ isolates had similar sequences, which clustered as a novel Helicobacter sp. closely related to H. mustelae (96-97%). The genome sequence of isolate MIT 01-6242 was assembled into a single ~1.6 Mb long contig with a 40.8% G+C content. The annotated genome contained 1699 protein-coding sequences and 43 RNAs, including 65 genes homologous to known Helicobacter spp. and Campylobacter spp. virulence factors. Histological changes in the gastric tissues extended from mild cystic degeneration of gastric glands to severe mucosal erosions and ulcers. Silver stains of infected tissues demonstrated slightly curved bacterial rods at the periphery of the gastric ulcers and on the epithelial surface of glands. The underlying mucosa and submucosa were infiltrated by low numbers of neutrophils, macrophages, and lymphocytes, with occasional lymphoid aggregates and well-defined lymphoid follicles. This is the second novel Helicobacter sp., which we have named ‘H. enhydrae’, isolated from inflamed stomachs of mustelids, the first being H. mustelae from a ferret.

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July 7, 2019

Beyond speciation genes: an overview of genome stability in evolution and speciation.

Genome stability ensures individual fitness and reliable transmission of genetic information. Hybridization between diverging lineages can trigger genome instability, highlighting its potential role in post-zygotic reproductive isolation. We argue that genome instability is not merely one of several types of hybrid incompatibility, but rather that genome stability is one of the very first and most fundamental traits that can break down when two diverged genomes are combined. Future work will reveal how frequent and predictable genome instability is in hybrids, how it affects hybrid fitness, and whether it is a direct cause or consequence of speciation. Copyright © 2017 Elsevier Ltd. All rights reserved.

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July 7, 2019

Comparative genomic analysis identifies a Campylobacter clade deficient in selenium metabolism.

The nonthermotolerant Campylobacter species C. fetus, C. hyointestinalis, C. iguaniorum, and C. lanienae form a distinct phylogenetic cluster within the genus. These species are primarily isolated from foraging (swine) or grazing (e.g., cattle, sheep) animals and cause sporadic and infrequent human illness. Previous typing studies identified three putative novel C. lanienae-related taxa, based on either MLST or atpA sequence data. To further characterize these putative novel taxa and the C. fetus group as a whole, 76 genomes were sequenced, either to completion or to draft level. These genomes represent 26 C. lanienae strains and 50 strains of the three novel taxa. C. fetus, C. hyointestinalis and C. iguaniorum genomes were previously sequenced to completion; therefore, a comparative genomic analysis across the entire C. fetus group was conducted (including average nucleotide identity analysis) that supports the initial identification of these three novel Campylobacter species. Furthermore, C. lanienae and the three putative novel species form a discrete clade within the C. fetus group, which we have termed the C. lanienae clade. This clade is distinguished from other members of the C. fetus group by a reduced genome size and distinct CRISPR/Cas systems. Moreover, there are two signature characteristics of the C. lanienae clade. C. lanienae clade genomes carry four to ten unlinked and similar, but nonidentical, flagellin genes. Additionally, all 76 C. lanienae clade genomes sequenced demonstrate a complete absence of genes related to selenium metabolism, including genes encoding the selenocysteine insertion machinery, selenoproteins, and the selenocysteinyl tRNA. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution 2017. This work is written by US Government employees and is in the public domain in the US.

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July 7, 2019

Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

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July 7, 2019

Complete genome sequence of the nematicidal Bacillus thuringiensis MYBT18246.

Bacillus thuringiensis is a rod-shaped facultative anaerobic spore forming bacterium of the genus Bacillus . The defining feature of the species is the ability to produce parasporal crystal inclusion bodies, consisting of d-endotoxins, encoded by cry-genes. Here we present the complete annotated genome sequence of the nematicidal B. thuringiensis strain MYBT18246. The genome comprises one 5,867,749 bp chromosome and 11 plasmids which vary in size from 6330 bp to 150,790 bp. The chromosome contains 6092 protein-coding and 150 RNA genes, including 36 rRNA genes. The plasmids encode 997 proteins and 4 t-RNA’s. Analysis of the genome revealed a large number of mobile elements involved in genome plasticity including 11 plasmids and 16 chromosomal prophages. Three different nematicidal toxin genes were identified and classified according to the Cry toxin naming committee as cry13Aa2, cry13Ba1, and cry13Ab1. Strikingly, these genes are located on the chromosome in close proximity to three separate prophages. Moreover, four putative toxin genes of different toxin classes were identified on the plasmids p120510 (Vip-like toxin), p120416 (Cry-like toxin) and p109822 (two Bin-like toxins). A comparative genome analysis of B. thuringiensis MYBT18246 with three closely related B. thuringiensis strains enabled determination of the pan-genome of B. thuringiensis MYBT18246, revealing a large number of singletons, mostly represented by phage genes, morons and cryptic genes.

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July 7, 2019

Paenibacillus ihbetae sp. nov., a cold-adapted antimicrobial producing bacterium isolated from high altitude Suraj Tal Lake in the Indian trans-Himalayas.

The assessment of bacterial diversity and bioprospection of the high-altitude lake Suraj Tal microorganisms for potent antimicrobial activities revealed the presence of two Gram-stain-variable, endospore-forming, rod-shaped, aerobic bacteria, namely IHBB 9852(T) and IHBB 9951. Phylogenetic analysis based on 16S rRNA gene sequence showed the affiliation of strains IHBB 9852(T) and IHBB 9951 within the genus Paenibacillus, exhibiting the highest sequence similarity to Paenibacillus lactis DSM 15596(T) (97.8% and 97.7%) and less than 95.9% similarity to other species of the genus Paenibacillus. DNA-DNA relatedness among strains IHBB 9852(T) and IHBB 9951 was 90.2%, and with P. lactis DSM 15596(T), was 52.7% and 52.4%, respectively. The novel strains contain anteiso-C15:0, iso-C15:0, C16:0 and iso-C16:0 as major fatty acids, and phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol were predominant polar lipids. The DNA G+C content for IHBB 9852T and IHBB 9951 was 52.1 and 52.2mol%. Based on the results of phenotypic and genomic characterisations, we concluded that strains IHBB 9852(T) and IHBB 9951 belong to a novel Paenibacillus species, for which the name Paenibacillus ihbetae sp. nov. is proposed. The type strain is IHBB 9852(T) (=MTCC 12459(T)=MCC 2795(T)=JCM 31131(T)=KACC 19072(T); DPD TaxonNumber TA00046) and IHBB 9951 (=MTCC 12458=MCC 2794=JCM 31132=KACC 19073) is a reference strain. Copyright © 2017. Published by Elsevier GmbH.

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July 7, 2019

Towards systems metabolic engineering in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is firmly established as a host for the production of recombinant proteins, frequently outperforming other heterologous hosts. Already, a sizeable amount of systems biology knowledge has been acquired for this non-conventional yeast. By applying various omics-technologies, productivity features have been thoroughly analyzed and optimized via genetic engineering. However, challenging clonal variability, limited vector repertoire and insufficient genome annotation have hampered further developments. Yet, in the last few years a reinvigorated effort to establish P. pastoris as a host for both protein and metabolite production is visible. A variety of compounds from terpenoids to polyketides have been synthesized, often exceeding the productivity of other microbial systems. The clonal variability was systematically investigated and strategies formulated to circumvent untargeted events, thereby streamlining the screening procedure. Promoters with novel regulatory properties were discovered or engineered from existing ones. The genetic tractability was increased via the transfer of popular manipulation and assembly techniques, as well as the creation of new ones. A second generation of sequencing projects culminated in the creation of the second best functionally annotated yeast genome. In combination with landmark physiological insights and increased output of omics-data, a good basis for the creation of refined genome-scale metabolic models was created. The first application of model-based metabolic engineering in P. pastoris showcased the potential of this approach. Recent efforts to establish yeast peroxisomes for compartmentalized metabolite synthesis appear to fit ideally with the well-studied high capacity peroxisomal machinery of P. pastoris. Here, these recent developments are collected and reviewed with the aim of supporting the establishment of systems metabolic engineering in P. pastoris. Copyright © 2017. Published by Elsevier Inc.

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July 7, 2019

Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism’s physiology, metabolism, and pathogenesis in human disease.

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July 7, 2019

From isolate to answer: how whole genome sequencing is helping us rapidly characterise nosocomial bacterial outbreaks

The occurrence of highly resistant bacterial pathogens has risen in recent years, causing immense strain on the healthcare industry. Hospital-acquired infections are arguably of most concern, as bacterial outbreaks in clinical settings provide an ideal environment for proliferation among vulnerable populations. Understanding these outbreaks beyond what can be determined with traditional clinical diagnostics and implementing these new techniques routinely in the hospital environment has now become a major focus. This brief review will discuss the three main whole genome sequence techniques available today, and how they are being used to further discriminate bacterial outbreaks in nosocomial settings.

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July 7, 2019

Centrochromatin of fungi.

The centromere is an essential chromosomal locus that dictates the nucleation point for assembly of the kinetochore and subsequent attachment of spindle microtubules during chromosome segregation. Research over the last decades demonstrated that centromeres are defined by a combination of genetic and epigenetic factors. Recent work showed that centromeres are quite diverse and flexible and that many types of centromere sequences and centromeric chromatin (“centrochromatin”) have evolved. The kingdom of the fungi serves as an outstanding example of centromere plasticity, including organisms with centromeres as diverse as 0.15-300 kb in length, and with different types of chromatin states for most species examined thus far. Some of the species in the less familiar taxa provide excellent opportunities to help us better understand centromere biology in all eukaryotes, which may improve treatment options against fungal infection, and biotechnologies based on fungi. This review summarizes the current knowledge of fungal centromeres and centrochromatin, including an outlook for future research.

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July 7, 2019

Whole-genome de novo sequencing of the lignin-degrading wood rot fungus Phanerochaete chrysosporium (ATCC 20696).

Phanerochaete chrysosporium (ATCC 20696) has a catabolic ability to degrade lignin. Here, we report whole-genome sequencing used to identify genes related to lignin modification. We determined the 39-Mb draft genome sequence of this fungus, comprising 13,560 predicted gene models. Gene annotation provided crucial information about the location and function of protein-encoding genes. Copyright © 2017 Hong et al.

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July 7, 2019

The Apostasia genome and the evolution of orchids.

Constituting approximately 10% of flowering plant species, orchids (Orchidaceae) display unique flower morphologies, possess an extraordinary diversity in lifestyle, and have successfully colonized almost every habitat on Earth. Here we report the draft genome sequence of Apostasia shenzhenica, a representative of one of two genera that form a sister lineage to the rest of the Orchidaceae, providing a reference for inferring the genome content and structure of the most recent common ancestor of all extant orchids and improving our understanding of their origins and evolution. In addition, we present transcriptome data for representatives of Vanilloideae, Cypripedioideae and Orchidoideae, and novel third-generation genome data for two species of Epidendroideae, covering all five orchid subfamilies. A. shenzhenica shows clear evidence of a whole-genome duplication, which is shared by all orchids and occurred shortly before their divergence. Comparisons between A. shenzhenica and other orchids and angiosperms also permitted the reconstruction of an ancestral orchid gene toolkit. We identify new gene families, gene family expansions and contractions, and changes within MADS-box gene classes, which control a diverse suite of developmental processes, during orchid evolution. This study sheds new light on the genetic mechanisms underpinning key orchid innovations, including the development of the labellum and gynostemium, pollinia, and seeds without endosperm, as well as the evolution of epiphytism; reveals relationships between the Orchidaceae subfamilies; and helps clarify the evolutionary history of orchids within the angiosperms.

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July 7, 2019

Identification of novel conjugative plasmids with multiple copies of fosB that confer high-level fosfomycin resistance to vancomycin-resistant Enterococci.

To further characterize the fosB-carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the fosB- and vanA-containing plasmids of Enterococcus faecium (pEMA120) and E. avium (pEA19081) were obtained by single-molecule, real-time sequencing. We found that these two plasmids are essentially identical (99.99% nucleotide sequence identity), which proved the possibility of interspecies transmission. Comparative analysis of the plasmids revealed that the backbone of pEMA120 is 99% similar to a conjugative fosB-negative E. faecium plasmid, pZB18. There is a traE disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact traE. The difference of their transfer frequencies between pEMA120 and pZB18 suggests this interruption of traE might affect conjugative transfer. Two copies of the fosB gene linked to a tnpA gene, forming an ISL3-like transposon, were found at separate locations within pEMA120, which had not been reported previously. These two fosB-carrying transposons were confirmed to form circular intermediates by inverse PCR. The hybridization of plasmid DNA digested by BsaI, having restriction site within the fosB sequence, demonstrated that the presence of multiple copies of fosB per plasmid is common. The total copy number of the fosB gene as revealed by qRT-PCR did not correlate with fosfomycin MICs or growth rates at sub-MICs of fosfomycin in different transconjugants. From susceptibility tests, the fosB gene, regardless of the copy number, conferred high fosfomycin MICs that ranged from 16384 to 65536 µg/ml. This first complete nucleotide sequence of a plasmid carrying two copies of fosB in VRE suggests that the fosB gene can transfer to multiple loci of plasmids by the ISL3 family transposase TnpA, possibly in the form of circular intermediates, leading to the dissemination of high fosfomycin resistance in VRE.

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July 7, 2019

The mobilome; A major contributor to Escherichia coli stx2-positive O26:H11 strains intra-serotype diversity.

Shiga toxin-producing Escherichia coli of serotype O26:H11/H- constitute a diverse group of strains and several clones with distinct genetic characteristics have been identified and characterized. Whole genome sequencing was performed using Illumina and PacBio technologies on eight stx2-positive O26:H11 strains circulating in France. Comparative analyses of the whole genome of the stx2-positive O26:H11 strains indicate that several clones of EHEC O26:H11 are co-circulating in France. Phylogenetic analysis of the French strains together with stx2-positive and stx-negative E. coli O26:H11 genomes obtained from Genbank indicates the existence of four clonal complexes (SNP-CCs) separated in two distinct lineages, one of which comprises the “new French clone” (SNP-CC1) that appears genetically closely related to stx-negative attaching and effacing E. coli (AEEC) strains. Interestingly, the whole genome SNP (wgSNP) phylogeny is summarized in the cas gene phylogeny, and a simple qPCR assay targeting the CRISPR array specific to SNP-CC1 (SP_O26-E) can distinguish between the two main lineages. The PacBio sequencing allowed a detailed analysis of the mobile genetic elements (MGEs) of the strains. Numerous MGEs were identified in each strain, including a large number of prophages and up to four large plasmids, representing overall 8.7-19.8% of the total genome size. Analysis of the prophage pool of the strains shows a considerable diversity with a complex history of recombination. Each clonal complex (SNP-CC) is characterized by a unique set of plasmids and phages, including stx-prophages, suggesting evolution through separate acquisition events. Overall, the MGEs appear to play a major role in O26:H11 intra-serotype clonal diversification.

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