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July 7, 2019

High-coverage sequencing and annotated assemblies of the budgerigar genome.

Parrots belong to a group of behaviorally advanced vertebrates and have an advanced ability of vocal learning relative to other vocal-learning birds. They can imitate human speech, synchronize their body movements to a rhythmic beat, and understand complex concepts of referential meaning to sounds. However, little is known about the genetics of these traits. Elucidating the genetic bases would require whole genome sequencing and a robust assembly of a parrot genome.We present a genomic resource for the budgerigar, an Australian Parakeet (Melopsittacus undulatus) — the most widely studied parrot species in neuroscience and behavior. We present genomic sequence data that includes over 300× raw read coverage from multiple sequencing technologies and chromosome optical maps from a single male animal. The reads and optical maps were used to create three hybrid assemblies representing some of the largest genomic scaffolds to date for a bird; two of which were annotated based on similarities to reference sets of non-redundant human, zebra finch and chicken proteins, and budgerigar transcriptome sequence assemblies. The sequence reads for this project were in part generated and used for both the Assemblathon 2 competition and the first de novo assembly of a giga-scale vertebrate genome utilizing PacBio single-molecule sequencing.Across several quality metrics, these budgerigar assemblies are comparable to or better than the chicken and zebra finch genome assemblies built from traditional Sanger sequencing reads, and are sufficient to analyze regions that are difficult to sequence and assemble, including those not yet assembled in prior bird genomes, and promoter regions of genes differentially regulated in vocal learning brain regions. This work provides valuable data and material for genome technology development and for investigating the genomics of complex behavioral traits.


July 7, 2019

Quorum sensing activity of Aeromonas caviae strain YL12, a bacterium isolated from compost.

Quorum sensing is a well-studied cell-to-cell communication method that involves a cell-density dependent regulation of genes expression mediated by signalling molecules. In this study, a bacterium isolated from a plant material compost pile was found to possess quorum sensing activity based on bioassay screening. Isolate YL12 was identified using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and molecular typing using rpoD gene which identified the isolate as Aeromonas caviae. High resolution tandem mass spectrometry was subsequently employed to identify the N-acyl homoserine lactone profile of Aeromonas caviae YL12 and confirmed that this isolate produced two short chain N-acyl homoserine lactones, namely C4-HSL and C6, and the production was observed to be cell density-dependent. Using the thin layer chromatography (TLC) bioassay, both AHLs were found to activate C. violaceum CV026, whereas only C6-HSL was revealed to induce bioluminescence expression of E. coli [pSB401]. The data presented in this study will be the leading steps in understanding the role of quorum sensing in Aeromonas caviae strain YL12.


July 7, 2019

Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria.

In recent years, the number of human infections caused by opportunistic pathogens has increased dramatically. Plant rhizospheres are one of the most typical natural reservoirs for these pathogens but they also represent a great source for beneficial microbes with potential for biotechnological applications. However, understanding the natural variation and possible differences between pathogens and beneficials is the main challenge in furthering these possibilities. The genus Stenotrophomonas contains representatives found to be associated with human and plant host.We used comparative genomics as well as transcriptomic and physiological approaches to detect significant borders between the Stenotrophomonas strains: the multi-drug resistant pathogenic S. maltophilia and the plant-associated strains S. maltophilia R551-3 and S. rhizophila DSM14405T (both are biocontrol agents). We found an overall high degree of sequence similarity between the genomes of all three strains. Despite the notable similarity in potential factors responsible for host invasion and antibiotic resistance, other factors including several crucial virulence factors and heat shock proteins were absent in the plant-associated DSM14405T. Instead, S. rhizophila DSM14405T possessed unique genes for the synthesis and transport of the plant-protective spermidine, plant cell-wall degrading enzymes, and high salinity tolerance. Moreover, the presence or absence of bacterial growth at 37°C was identified as a very simple method in differentiating between pathogenic and non-pathogenic isolates. DSM14405T is not able to grow at this human-relevant temperature, most likely in great part due to the absence of heat shock genes and perhaps also because of the up-regulation at increased temperatures of several genes involved in a suicide mechanism.While this study is important for understanding the mechanisms behind the emerging pattern of infectious diseases, it is, to our knowledge, the first of its kind to assess the risk of beneficial strains for biotechnological applications. We identified certain traits typical of pathogens such as growth at the human body temperature together with the production of heat shock proteins as opposed to a temperature-regulated suicide system that is harnessed by beneficials.


July 7, 2019

The Glanville fritillary genome retains an ancient karyotype and reveals selective chromosomal fusions in Lepidoptera.

Previous studies have reported that chromosome synteny in Lepidoptera has been well conserved, yet the number of haploid chromosomes varies widely from 5 to 223. Here we report the genome (393?Mb) of the Glanville fritillary butterfly (Melitaea cinxia; Nymphalidae), a widely recognized model species in metapopulation biology and eco-evolutionary research, which has the putative ancestral karyotype of n=31. Using a phylogenetic analyses of Nymphalidae and of other Lepidoptera, combined with orthologue-level comparisons of chromosomes, we conclude that the ancestral lepidopteran karyotype has been n=31 for at least 140?My. We show that fusion chromosomes have retained the ancestral chromosome segments and very few rearrangements have occurred across the fusion sites. The same, shortest ancestral chromosomes have independently participated in fusion events in species with smaller karyotypes. The short chromosomes have higher rearrangement rate than long ones. These characteristics highlight distinctive features of the evolutionary dynamics of butterflies and moths.


July 7, 2019

Whole-genome sequence of Burkholderia sp. strain RPE67, a bacterial gut symbiont of the bean bug Riptortus pedestris.

Burkholderia sp. strain RPE67 is a bacterial symbiont isolated from a field-collected bean bug, Riptortus pedestris. To understand the genetic basis of the insect-microbe symbiosis, we performed whole-genome sequencing of the Burkholderia strain, revealing an 8.69-Mb genome consisting of three chromosomes and three plasmids. Copyright © 2014 Takeshita et al.


July 7, 2019

Complete genome sequence of Kosakonia sacchari type strain SP1(T.).

Kosakonia sacchari sp. nov. is a new species within the new genus Kosakonia, which was included in the genus Enterobacter. K sacchari is a nitrogen-fixing bacterium named for its association with sugarcane (Saccharum officinarum L.). K sacchari bacteria are Gram-negative, aerobic, non-spore-forming, motile rods. Strain SP1(T) (=CGMCC1.12102(T)=LMG 26783(T)) is the type strain of the K sacchari sp. nov and is able to colonize and fix N2 in association with sugarcane plants, thus promoting plant growth. Here we summarize the features of strain SP1(T) and describe its complete genome sequence. The genome contains a single chromosome and no plasmids, 4,902,024 nucleotides with 53.7% GC content, 4,460 protein-coding genes and 105 RNA genes including 22 rRNA genes, 82 tRNA genes, and 1 ncRNA gene.


July 7, 2019

Complete genome sequences of Salmonella enterica serovar Heidelberg strains associated with a multistate food-borne illness investigation.

Next-generation sequencing is being evaluated for use with food-borne illness investigations, especially when the outbreak strains produce patterns that cannot be discriminated from non-outbreak strains using conventional procedures. Here we report complete genome assemblies of two Salmonella enterica serovar Heidelberg strains with a common pulsed-field gel electrophoresis pattern isolated during an outbreak investigation.


July 7, 2019

Site-specific genetic engineering of the Anopheles gambiae Y chromosome.

Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line-specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.


July 7, 2019

Molecular and biological characterization of a new isolate of guinea pig cytomegalovirus.

Development of a vaccine against congenital infection with human cytomegalovirus is complicated by the issue of re-infection, with subsequent vertical transmission, in women with pre-conception immunity to the virus. The study of experimental therapeutic prevention of re-infection would ideally be undertaken in a small animal model, such as the guinea pig cytomegalovirus (GPCMV) model, prior to human clinical trials. However, the ability to model re-infection in the GPCMV model has been limited by availability of only one strain of virus, the 22122 strain, isolated in 1957. In this report, we describe the isolation of a new GPCMV strain, the CIDMTR strain. This strain demonstrated morphological characteristics of a typical Herpesvirinae by electron microscopy. Illumina and PacBio sequencing demonstrated a genome of 232,778 nt. Novel open reading frames ORFs not found in reference strain 22122 included an additional MHC Class I homolog near the right genome terminus. The CIDMTR strain was capable of dissemination in immune compromised guinea pigs, and was found to be capable of congenital transmission in GPCMV-immune dams previously infected with salivary gland-adapted strain 22122 virus. The availability of a new GPCMV strain should facilitate study of re-infection in this small animal model.


July 7, 2019

The effects of read length, quality and quantity on microsatellite discovery and primer development: from Illumina to PacBio.

The advent of next-generation sequencing (NGS) technologies has transformed the way microsatellites are isolated for ecological and evolutionary investigations. Recent attempts to employ NGS for microsatellite discovery have used the 454, Illumina, and Ion Torrent platforms, but other methods including single-molecule real-time DNA sequencing (Pacific Biosciences or PacBio) remain viable alternatives. We outline a workflow from sequence quality control to microsatellite marker validation in three plant species using PacBio circular consensus sequencing (CCS). We then evaluate the performance of PacBio CCS in comparison with other NGS platforms for microsatellite isolation, through simulations that focus on variations in read length, read quantity and sequencing error rate. Although quality control of CCS reads reduced microsatellite yield by around 50%, hundreds of microsatellite loci that are expected to have improved conversion efficiency to functional markers were retrieved for each species. The simulations quantitatively validate the advantages of long reads and emphasize the detrimental effects of sequencing errors on NGS-enabled microsatellite development. In view of the continuing improvement in read length on NGS platforms, sequence quality and the corresponding strategies of quality control will become the primary factors to consider for effective microsatellite isolation. Among current options, PacBio CCS may be optimal for rapid, small-scale microsatellite development due to its flexibility in scaling sequencing effort, while platforms such as Illumina MiSeq will provide cost-efficient solutions for multispecies microsatellite projects. © 2014 John Wiley & Sons Ltd.


July 7, 2019

Complete closed genome sequences of three Bibersteinia trehalosi nasopharyngeal isolates from cattle with shipping fever.

Bibersteinia trehalosi is a respiratory pathogen affecting cattle and related ruminants worldwide. B. trehalosi is closely related to Mannheimia haemolytica and is often associated with bovine respiratory disease complex (BRDC), a polymicrobial multifactorial disease. We present three complete closed genome sequences of this species generated using an automated assembly pipeline.


July 7, 2019

The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

The facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic ß-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway.Castellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate.The genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.


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