Fire blight, caused by the enterobacterium Erwinia amylovora, is a devastating disease of rosaceous plants that has global economic importance for apple and pear production and trade. The complete genome of E. amylovora CFBP 1430 was sequenced, annotated, and compared with the genomes of other Erwinia spp. Several singleton and shared features of the E. amylovora CFBP 1430 genome were identified that offer a first view into evolutionary aspects within the genus Erwinia. Comparative genomics identified or clarified virulence and fitness determinants and secretion systems. Novel insights revealed in the genome of E. amylovora CFBP 1430 hold potential for exploitation to improve the design of more effective fire blight control strategies.

Accurate annotation of all protein-coding sequences (CDSs) is an essential prerequisite to fully exploit the rapidly growing repertoire of completely sequenced prokaryotic genomes. However, large discrepancies among the number of CDSs annotated by different resources, missed functional short open reading frames (sORFs), and overprediction of spurious ORFs represent serious limitations. Our strategy toward accurate and complete genome annotation consolidates CDSs from multiple reference annotation resources, ab initio gene prediction algorithms and in silico ORFs (a modified six-frame translation considering alternative start codons) in an integrated proteogenomics database (iPtgxDB) that covers the entire proteincoding potential of a prokaryotic genome. By extending the PeptideClassifier concept of unambiguous peptides for prokaryotes, close to 95% of the identifiable peptides imply one distinct protein, largely simplifying downstream analysis. Searching a comprehensive Bartonella henselae proteomics data set against such an iPtgxDB allowed us to unambiguously identify novel ORFs uniquely predicted by each resource, including lipoproteins, differentially expressed and membrane-localized proteins, novel start sites and wrongly annotated pseudogenes. Most novelties were confirmed by targeted, parallel reaction monitoring mass spectrometry, including unique ORFs and single amino acid variations (SAAVs) identified in a re-sequenced laboratory strain that are not present in its reference genome. We demonstrate the general applicability of our strategy for genomes with varying GC content and distinct taxonomic origin. We release iPtgxDBs for B. henselae, Bradyrhizobium diazoefficiens and Escherichia coli and the software to generate both proteogenomics search databases and integrated annotation files that can be viewed in a genome browser for any prokaryote.

Neonicotinoids are implicated in the decline of honey bees, but the molecular basis underlying adverse effects is poorly known. Here we describe global transcriptomic profiles in the brain of honey bee workers exposed for 48 h at one environmentally realistic and one sublethal concentration of 0.3 and 3.0 ng/bee clothianidin and imidacloprid, respectively, and 0.1 and 1.0 ng/bee thiamethoxam (1-30 ng/mL sucrose solution) by high-throughput RNA-sequencing (RNA-seq). All neonicotinoids led to significant alteration (mainly down-regulation) of gene expression, generally with a concentration-dependent effect. Among many others, genes related to metabolism and detoxification were differently expressed. Gene ontology (GO) enrichment analysis of biological processes revealed catabolic carbohydrate metabolism (regulation of enzyme activities such as amylase), lipid metabolism, and transport mechanisms as shared terms between all neonicotinoids at high concentrations. KEGG pathway analysis indicated that at least two neonicotinoids induced changes in expression of various metabolic pathways: pentose phosphate pathways, starch and sucrose metabolism, and sulfur metabolism, in which glucose 1-dehydrogenase and alpha-amylase were down-regulated and 3'(2'), 5'-bisphosphate nucleotidase was up-regulated. RT-qPCR analysis confirmed the down-regulation of major royal jelly proteins, hbg3, and cyp9e2 found by RNA-seq. Our study highlights the comparative molecular effects of neonicotinoid exposure to bees. Further studies should link these effects with physiological outcomes for a better understanding of effects of neonicotinoids.

Pantoea vagans is a Gram-negative enterobacterial plant epiphyte of a broad range of plants. Here we report the 4.89-Mb genome sequence of P. vagans strain C9-1 (formerly Pantoea agglomerans), which is commercially registered for biological control of fire blight, a disease of pear and apple trees caused by Erwinia amylovora.Pantoea vagans (syn. Pantoea agglomerans, Erwinia herbicola) (10), is a common plant epiphyte. Strain C9-1, isolated from apple (Malus ϫ domestica 'Jonathan') (Michigan) (5), is registered as BlightBan C9-1 (Nufarms America Inc., Burr Ridge, IL) for biocontrol of fire blight caused by the related enterobacterium Erwinia amylovora. Applied during bloom, C9-1 provides effective disease control, similar to oxytetracycline and slightly less disease control than streptomycin treatment provides (6). As for most biocontrol agents, growth and efficacy vary among locations and years (6). We sequenced the complete genome of P. vagans C9-1 as a step toward elucidating genetic factors that modulate performance reliability.Genomic DNA, isolated using the Wizard genomic DNA purification kit (Promega, Madison, WI) was whole-genome shot-gun sequenced by 454 Life Sciences (Branford, CT) with four runs on a GS-20 sequencer. This resulted in 1,224,924 high-quality filtered reads with an average read length of 97 bp and coverage equivalent to 25 times. Quality filtered sequences were assembled in silico using the 454 Newbler assembler giving 207 contigs, 43 contigs larger than 500 bp. Gap closure and repetitive element sequencing were achieved by PCR walking using the Sanger method on a 3130XL sequencer (Applied Biosystems, Rotkreuz, Switzerland). Final assembly and sequence manipulations were done using the Lasergene package v8.1 (DNASTAR, Madison, WI).The P. vagans C9-1 genome has a 4,025-Mb circular chromosome and three circular plasmids (pPag1 [167,983 bp], pPag2 [165,692 bp], and pPag3 [529,676 bp]). A total of 4,619 coding sequences were predicted (8) using GLIMMER (11) and CRITICA (1). Putative functions of protein-encoding genes were automatically assigned using the GenDB annotation pipeline (9) and manually optimized. The chromosome harbors 7 rRNA operons and 78 tRNAs over the chromosome.The P. vagans C9-1 genome has four large regions containing phage-related genes. Bordering regions could not be identified explicitly (e.g., one phage region is flanked by a tRNA Met[AttL] and an AttR region, while others were on the other side of AttR). Transposon-related genes were largely absent, but small genomic islands were observed. Only plasmid pPag2 contains remnants of transposases and recombinases, including an inactivated ISEhe3 and ISEhe4, related to those on pPATH pag in P. agglomerans pv. gypsophilae 824-1 (4).The P. vagans C9-1 genome contains a repertoire of carbohydrate metabolic pathways identified using the KEGG databases (7). Important biosynthetic genes for antibacterial metabolites (i.e., pantocin A and dapdiamide E) and epiphytic fitness genes (e.g., autoinducer 1 [AI-1] quorum-sensing genes, in...

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BackgroundRapid and reliable identification of quarantine pests is essential for plant inspection services to prevent introduction of invasive species. For insects, this may be a serious problem when dealing with morphologically similar cryptic species complexes and early developmental stages that lack distinctive characters useful for taxonomic identification. DNA based barcoding could solve many of these problems. The standard barcode fragment, an approx. 650 base pairs long sequence of the 5′end of the mitochondrial cytochrome oxidase I (COI), enables differentiation of a very wide range of arthropods. However, problems remain in some taxa, such as Tephritidae, where recent genetic differentiation among some of the described species hinders accurate molecular discrimination.ResultsIn order to explore the full species discrimination potential of COI, we sequenced the barcoding region of the COI gene of a range of economically important Tephritid species and complemented these data with all GenBank and BOLD entries for the systematic group available as of January 2012. We explored the limits of species delimitation of this barcode fragment among 193 putative Tephritid species and established operational taxonomic units (OTUs), between which discrimination is reliably possible. Furthermore, to enable future development of rapid diagnostic assays based on this sequence information, we characterized all single nucleotide polymorphisms (SNPs) and established “near-minimal” sets of SNPs that differentiate among all included OTUs with at least three and four SNPs, respectively.ConclusionsWe found that although several species cannot be differentiated based on the genetic diversity observed in COI and hence form composite OTUs, 85% of all OTUs correspond to described species. Because our SNP panels are developed based on all currently available sequence information and rely on a minimal pairwise difference of three SNPs, they are highly reliable and hence represent an important resource for developing taxon-specific diagnostic assays. For selected cases, possible explanations that may cause composite OTUs are discussed.

Background Pantoea vagans is a commercialized biological control agent used against the pome fruit bacterial disease fire blight, caused by Erwinia amylovora. Compared to other biocontrol agents, relatively little is currently known regarding Pantoea genetics. Better understanding of antagonist mechanisms of action and ecological fitness is critical to improving efficacy.Principal FindingsGenome analysis indicated two major factors contribute to biocontrol activity: competition for limiting substrates and antibacterial metabolite production. Pathways for utilization of a broad diversity of sugars and acquisition of iron were identified. Metabolism of sorbitol by P. vagans C9-1 may be a major metabolic feature in biocontrol of fire blight. Biosynthetic genes for the antibacterial peptide pantocin A were found on a chromosomal 28-kb genomic island, and for dapdiamide E on the plasmid pPag2. There was no evidence of potential virulence factors that could enable an animal or phytopathogenic lifestyle and no indication of any genetic-based biosafety risk in the antagonist.ConclusionsIdentifying key determinants contributing to disease suppression allows the development of procedures to follow their expression in planta and the genome sequence contributes to rationale risk assessment regarding the use of the biocontrol strain in agricultural systems.

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences' long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus—to our knowledge—identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.