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July 7, 2019

Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms.

CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two separate genes. Sequencing of three overlapping bacterial artificial chromosomes (BACs) spanning the entire canine CD1 locus revealed canCD1A8.2 and canCD1A8.1 to be located in tandem between canCD1A7 and canCD1C, and canCD1A8.1 was consequently renamed canCD1A9. Green fluorescent protein (GFP)-fused canine CD1 transcripts were recombinantly expressed in 293T cells. All proteins showed a highly positive GFP expression except for canine CD1d and a splice variant of canine CD1a8 lacking exon 3. Probing with a panel of anti-CD1 monoclonal antibodies (mAbs) showed that Ca13.9H11 and Ca9.AG5 only recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our findings have refined the structure of the canine CD1 locus and available antibody specificity against canine CD1 proteins. These are important fundamentals for future investigation of the role of canine CD1 in lipid immunity.

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July 7, 2019

Plasmid characterization and chromosome analysis of two netF+ Clostridium perfringens isolates associated with foal and canine necrotizing enteritis.

The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus.

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July 7, 2019

Complete genome sequence of the potato pathogen Ralstonia solanacearum UY031.

Ralstonia solanacearum is the causative agent of bacterial wilt of potato. Ralstonia solanacearum strain UY031 belongs to the American phylotype IIB, sequevar 1, also classified as race 3 biovar 2. Here we report the completely sequenced genome of this strain, the first complete genome for phylotype IIB, sequevar 1, and the fourth for the R. solanacearum species complex. In addition to standard genome annotation, we have carried out a curated annotation of type III effector genes, an important pathogenicity-related class of genes for this organism. We identified 60 effector genes, and observed that this effector repertoire is distinct when compared to those from other phylotype IIB strains. Eleven of the effectors appear to be nonfunctional due to disruptive mutations. We also report a methylome analysis of this genome, the first for a R. solanacearum strain. This analysis helped us note the presence of a toxin gene within a region of probable phage origin, raising the hypothesis that this gene may play a role in this strain’s virulence.

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July 7, 2019

Multiple and diverse vsp and vlp sequences in Borrelia miyamotoi, a hard tick-borne zoonotic pathogen.

Based on chromosome sequences, the human pathogen Borrelia miyamotoi phylogenetically clusters with species that cause relapsing fever. But atypically for relapsing fever agents, B. miyamotoi is transmitted not by soft ticks but by hard ticks, which also are vectors of Lyme disease Borrelia species. To further assess the relationships of B. miyamotoi to species that cause relapsing fever, I investigated extrachromosomal sequences of a North American strain with specific attention on plasmid-borne vsp and vlp genes, which are the underpinnings of antigenic variation during relapsing fever. For a hybrid approach to achieve assemblies that spanned more than one of the paralogous vsp and vlp genes, a database of short-reads from next-generation sequencing was supplemented with long-reads obtained with real-time DNA sequencing from single polymerase molecules. This yielded three contigs of 31, 16, and 11 kb, which each contained multiple and diverse sequences that were homologous to vsp and vlp genes of the relapsing fever agent B. hermsii. Two plasmid fragments had coding sequences for plasmid partition proteins that differed from each other from paralogous proteins for the megaplasmid and a small plasmid of B. miyamotoi. One of 4 vsp genes, vsp1, was present at two loci, one of which was downstream of a candiate prokaryotic promoter. A limited RNA-seq analysis of a population growing in the blood of mice indicated that of the 4 different vsp genes vsp1 was the one that was expressed. The findings indicate that B. miyamotoi has at least four types of plasmids, two or more of which bear vsp and vlp gene sequences that are as numerous and diverse as those of relapsing fever Borrelia. The database and insights from these findings provide a foundation for further investigations of the immune responses to this pathogen and of the capability of B. miyamotoi for antigenic variation.

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July 7, 2019

Genomes and virulence difference between two physiological races of Phytophthora nicotianae.

Black shank is a severe plant disease caused by the soil-borne pathogen Phytophthora nicotianae. Two physiological races of P. nicotianae, races 0 and 1, are predominantly observed in cultivated tobacco fields around the world. Race 0 has been reported to be more aggressive, having a shorter incubation period, and causing worse root rot symptoms, while race 1 causes more severe necrosis. The molecular mechanisms underlying the difference in virulence between race 0 and 1 remain elusive.We assembled and annotated the genomes of P. nicotianae races 0 and 1, which were obtained by a combination of PacBio single-molecular real-time sequencing and second-generation sequencing (both HiSeq and MiSeq platforms). Gene family analysis revealed a highly expanded ATP-binding cassette transporter gene family in P. nicotianae. Specifically, more RxLR effector genes were found in the genome of race 0 than in that of race 1. In addition, RxLR effector genes were found to be mainly distributed in gene-sparse, repeat-rich regions of the P. nicotianae genome.These results provide not only high quality reference genomes of P. nicotianae, but also insights into the infection mechanisms of P. nicotianae and its co-evolution with the host plant. They also reveal insights into the difference in virulence between the two physiological races.

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July 7, 2019

Draft genome sequence of Alternaria alternata ATCC 34957.

We report the draft genome sequence of Alternaria alternata ATCC 34957. This strain was previously reported to produce alternariol and alternariol monomethyl ether on weathered grain sorghum. The genome was sequenced with PacBio technology and assembled into 27 scaffolds with a total genome size of 33.5 Mb. Copyright © 2016 Nguyen et al.

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July 7, 2019

Transcriptional profiling the 150 kb linear megaplasmid of Borrelia turicatae suggests a role in vector colonization and initiating mammalian infection.

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3′ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

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July 7, 2019

Genome sequence of Nitrosomonas communis strain Nm2, a mesophilic ammonia-oxidizing bacterium isolated from Mediterranean soil.

The complete genome sequence of Nitrosomonas communis strain Nm2, a mesophilic betaproteobacterial ammonia oxidizer isolated from Mediterranean soils in Corfu, Greece, is reported here. This is the first genome to describe a cluster 8 Nitrosomonas species and represents an ammonia-oxidizing bacterium commonly found in terrestrial ecosystems. Copyright © 2016 Kozlowski et al.

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July 7, 2019

Complete genome sequence and genomic characterization of Microcystis panniformis FACHB 1757 by third-generation sequencing.

The cyanobacterial genus Microcystis is well known as the main group that forms harmful blooms in water. A strain of Microcystis, M. panniformis FACHB1757, was isolated from Meiliang Bay of Lake Taihu in August 2011. The whole genome was sequenced using PacBio RS II sequencer with 48-fold coverage. The complete genome sequence with no gaps contained a 5,686,839 bp chromosome and a 38,683 bp plasmid, which coded for 6,519 and 49 proteins, respectively. Comparison with strains of M. aeruginosa and some other water bloom-forming cyanobacterial species revealed large-scale structure rearrangement and length variation at the genome level along with 36 genomic islands annotated genome-wide, which demonstrates high plasticity of the M. panniformis FACHB1757 genome and reveals that Microcystis has a flexible genome evolution.

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July 7, 2019

Phylogenomic analysis reveals genome-wide purifying selection on TBE transposons in the ciliate Oxytricha.

Transposable elements are a major player contributing to genetic variation and shaping genome evolution. Multiple independent transposon domestication events have occurred in ciliates, recruiting transposases to key roles in cellular processes. In the ciliate Oxytricha trifallax, the telomere-bearing elements (TBE), a Tc1/mariner transposon, occupy a significant portion of the germline genome and are involved in programmed genome rearrangements that produce a transcriptionally active somatic nucleus from a copy of the germline nucleus during development.Here we provide a thorough characterization of the distribution and sequences of TBE transposons in the Oxytricha germline genome. We annotate more than 10,000 complete and 24,000 partial TBE sequences. TBEs cluster into four major families and display a preference for either insertion into DNA segments that are retained in the somatic genome or their maintenance at such sites. The three TBE-encoded genes in all four families display dN/dS ratios much lower than 1, suggesting genome-wide purifying selection. We also identify TBE homologs in other ciliate species for phylogenomic analysis.This paper provides genome-wide characterization of a major class of ciliate transposons. Phylogenomic analysis reveals selective constraints on transposon-encoded genes, shedding light on the evolution and domesticated functions of these transposons.

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July 7, 2019

Comparative analysis of an IncR plasmid carrying armA, blaDHA-1 and qnrB4 from Klebsiella pneumoniae ST37 isolates.

The objective of this study was to conduct a comparative analysis with reported IncR plasmids of a Klebsiella pneumoniae IncR plasmid carrying an MDR region.MDR K. pneumoniae isolates were serially identified from two inpatients at a hospital in the USA in 2014. MDR plasmid pYDC676 was fully sequenced, annotated and compared with related plasmids. Antimicrobial susceptibility testing, PFGE and MLST were also conducted.The K. pneumoniae isolates were identical by PFGE, belonged to ST37 and harboured an identical ~50 kb IncR plasmid (pYDC676). pYDC676 possessed the backbone and multi-IS loci closely related to IncR plasmids reported from aquatic bacteria, as well as animal and human K. pneumoniae strains, and carried an MDR region consisting of armA, blaDHA-1 and qnrB4, a combination that has been reported in IncR plasmids from K. pneumoniae ST11 strains in Europe and Asia. A plasmid with the identical IncR backbone and a similar MDR region containing blaDHA-1 and qnrB4 has also been reported in ST37 strains from Europe, suggesting potential dissemination of this lineage of IncR plasmids in K. pneumoniae ST37.K. pneumoniae ST37 strains with an MDR IncR plasmid carrying armA, blaDHA-1 and qnrB4 were identified in a hospital in the USA, where these resistance genes remain rare. The IncR backbone may play a role in the global dissemination of these resistance genes.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Genome of Cnaphalocrocis medinalis granulovirus, the first Crambidae-infecting betabaculovirus isolated from rice leaffolder to sequenced.

Cnaphalocrocis medinalis is a major pest of rice in South and South-East Asia. Insecticides are the major means farmers use for management. A naturally occurring baculovirus, C. medinalis granulovirus (CnmeGV), has been isolated from the larvae and this has the potential for use as microbial agent. Here, we described the complete genome sequence of CnmeGV and compared it to other baculovirus genomes. The genome of CnmeGV is 112,060 base pairs in length, has a G+C content of 35.2%. It contains 133 putative open reading frames (ORFs) of at least 150 nucleotides. A hundred and one (101) of these ORFs are homologous to other baculovirus genes including 37 baculovirus core genes. Thirty-two (32) ORFs are unique to CnmeGV with no homologues detected in the GeneBank and 53 tandem repeats (TRs) with sequence length from 25 to 551 nt intersperse throughout the genome of CnmeGV. Six (6) homologous regions (hrs) were identified interspersed throughout the genome. Hr2 contains 11 imperfect palindromes and a high content of AT sequence (about 73%). The unique ORF28 contains a coiled-coil region and a zinc finger-like domain of 4-50 residues specialized by two C2C2 zinc finger motifs that putatively bound two atoms of zinc. ORF21 encoding a chit-1 protein suggesting a horizontal gene transfer from alphabaculovirus. The putative protein presents two carbohydrate-binding module family 14 (CBM_14) domains rather than other homologues detected from betabaculovirus that only contains one chit-binding region. Gene synteny maps showed the colinearity of sequenced betabaculovirus. Phylogenetic analysis indicated that CnmeGV grouped in the betabaculovirus, with a close relation to AdorGV. The cladogram obtained in this work grouped the 17 complete GV genomes in one monophyletic clade. CnmeGV represents a new crambidae host-isolated virus species from the genus Betabaculovirus and is most closely relative of AdorGV. The analyses and information derived from this study will provide a better understanding of the pathological symptoms caused by this virus and its potential use as a microbial pesticide.

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