The symmetry spectrum in a hybridising, tropical group of rhododendrons.

Many diverse plant clades possess bilaterally symmetrical flowers and specialised pollination syndromes, suggesting that these traits may promote diversification. We examined the evolution of diverse floral morphologies in a species-rich tropical radiation of Rhododendron. We used restriction-site associated DNA sequencing on 114 taxa from Rhododendron sect. Schistanthe to reconstruct phylogenetic relationships and examine hybridisation. We then captured and quantified floral variation using geometric morphometric analyses, which we interpreted in a phylogenetic context. We uncovered phylogenetic conflict and uncertainty caused by introgression within and between clades. Morphometric analyses revealed flower symmetry to be a morphological continuum without clear transitions between radial and bilateral symmetry. Tropical Rhododendron species that began diversifying into New Guinea c. 6 million years ago expanded into novel floral morphological space. Our results showed that the evolution of tropical Rhododendron is characterised by recent speciation, recurrent hybridisation and the origin of floral novelty. Floral variation evolved via changes to multiple components of the corolla that are only recognised in geometric morphometrics with both front and side views of flowers.

Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii.

UNLABELLED: Acinetobacter baumannii is a Gram-negative bacterial pathogen notorious for causing serious nosocomial infections that resist antibiotic therapy. Research to identify factors responsible for the pathogen's success has been limited by the resources available for genome-scale experimental studies. This report describes the development of several such resources for A. baumannii strain AB5075, a recently characterized wound isolate that is multidrug resistant and displays robust virulence in animal models. We report the completion and annotation of the genome sequence, the construction of a comprehensive ordered transposon mutant library, the extension of high-coverage transposon mutant pool sequencing (Tn-seq) to the strain, and the identification of the genes essential for growth on nutrient-rich agar. These resources should facilitate large-scale genetic analysis of virulence, resistance, and other clinically relevant traits that make A. baumannii a formidable public health threat. IMPORTANCE: Acinetobacter baumannii is one of six bacterial pathogens primarily responsible for antibiotic-resistant infections that have become the scourge of health care facilities worldwide. Eliminating such infections requires a deeper understanding of the factors that enable the pathogen to persist in hospital environments, establish infections, and resist antibiotics. We present a set of resources that should accelerate genome-scale genetic characterization of these traits for a reference isolate of A. baumannii that is highly virulent and representative of current outbreak strains.

Sequence-verified two-allele transposon mutant library for Pseudomonas aeruginosa PAO1.

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.

Exploiting a natural auxotrophy for genetic selection.

We exploited the natural histidine auxotrophy of Francisella species to develop hisD (encodes histidinol dehydrogenase) as a positive selection marker. A shuttle plasmid (pBR103) carrying Escherichia coli hisD and designed for cloning of PCR fragments replicated in both attenuated and highly virulent Francisella strains. During this work, we formulated a simplified defined growth medium for Francisella novicida.

Gene Duplication and Differential Expression of Flower Symmetry Genes in Rhododendron (Ericaceae).

Bilaterally symmetric flowers have evolved over a hundred times in angiosperms, yet orthologs of the transcription factors CYCLOIDEA (CYC), RADIALIS (RAD), and DIVARICATA (DIV) are repeatedly implicated in floral symmetry changes. We examined these candidate genes to elucidate the genetic underpinnings of floral symmetry changes in florally diverse Rhododendron, reconstructing gene trees and comparing gene expression across floral organs in representative species with radial and bilateral flower symmetries. Radially symmetric R. taxifolium Merr. and bilaterally symmetric R. beyerinckianum Koord. had four and five CYC orthologs, respectively, from shared tandem duplications. CYC orthologs were expressed in the longer dorsal petals and stamens and highly expressed in R. beyerinckianum pistils, whereas they were either ubiquitously expressed, lost from the genome, or weakly expressed in R. taxifolium. Both species had two RAD and DIV orthologs uniformly expressed across all floral organs. Differences in gene structure and expression of Rhododendron RAD compared to other asterids suggest that these genes may not be regulated by CYC orthologs. Our evidence supports CYC orthologs as the primary regulators of differential organ growth in Rhododendron flowers, while also suggesting certain deviations from the typical asterid gene regulatory network for flower symmetry.

The Rhododendron Genome and Chromosomal Organization Provide Insight into Shared Whole-Genome Duplications across the Heath Family (Ericaceae).

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order.

Genetic dissection of the Francisella novicida restriction barrier.

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10(6)-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.

Creating recombination-activated genes and sequence-defined mutant libraries using transposons.

The properties of a collection of transposon Tn5 derivatives that generate reporter gene fusions and internal protein tags are summarized. Procedures utilizing several of the transposons for generating genes activated by Cre-loxP recombination and for creating large sequence-defined mutant libraries are described in detail.

In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075.

The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms.

Host-Microbe Protein Interactions during Bacterial Infection.

Interspecies protein-protein interactions are essential mediators of infection. While bacterial proteins required for host cell invasion and infection can be identified through bacterial mutant library screens, information about host target proteins and interspecies complex structures has been more difficult to acquire. Using an unbiased chemical crosslinking/mass spectrometry approach, we identified interspecies protein-protein interactions in human lung epithelial cells infected with Acinetobacter baumannii. These efforts resulted in identification of 3,076 crosslinked peptide pairs and 46 interspecies protein-protein interactions. Most notably, the key A. baumannii virulence factor, OmpA, was identified as crosslinked to host proteins involved in desmosomes, specialized structures that mediate host cell-to-cell adhesion. Co-immunoprecipitation and transposon mutant experiments were used to verify these interactions and demonstrate relevance for host cell invasion and acute murine lung infection. These results shed new light on A. baumannii-host protein interactions and their structural features, and the presented approach is generally applicable to other systems.

Sequence-defined transposon mutant library of Burkholderia thailandensis.

UNLABELLED: We constructed a near-saturation transposon mutant library for Burkholderia thailandensis, a low-virulence surrogate for the causative agent of melioidosis (Burkholderia pseudomallei). A primary set of nearly 42,000 unique mutants (~7.5 mutants/gene) was generated using transposon Tn5 derivatives. The strains carry insertions in 87% of the predicted protein-coding genes of the organism, corresponding to nearly all of those nonessential for growth on nutrient agar. To achieve high genome coverage, we developed procedures for efficient sequence identification of insertions in extremely GC-rich regions of DNA. To facilitate strain distribution, we created a secondary library with two mutants per gene for which most transposon locations had been confirmed by resequencing. A map of mutations in the two-allele library and procedures for obtaining strains can be found at http://tools.nwrce.org/tn_mutants/ and http://www.gs.washington.edu/labs/manoil/. The library should facilitate comprehensive mutant screens and serve as a source of strains to test predicted genotype-phenotype associations. IMPORTANCE: The Gram-negative bacterium Burkholderia pseudomallei is a biothreat agent due to its potential for aerosol delivery and intrinsic antibiotic resistance and because exposure produces pernicious infections. Large-scale studies of B. pseudomallei are limited by the fact that the organism must be manipulated under biological safety level 3 conditions. A close relative of B. pseudomallei called Burkholderia thailandensis, which can be studied under less restrictive conditions, has been validated as a low-virulence surrogate in studies of virulence, antibiotic resistance and other traits. To facilitate large-scale studies of B. thailandensis, we created a near-saturation, sequence-defined transposon mutant library of the organism. The library facilitates genetic studies that identify genotype-phenotype associations conserved in B. pseudomallei.

Evolution of Burkholderia pseudomallei in recurrent melioidosis.

Burkholderia pseudomallei, the etiologic agent of human melioidosis, is capable of causing severe acute infection with overwhelming septicemia leading to death. A high rate of recurrent disease occurs in adult patients, most often due to recrudescence of the initial infecting strain. Pathogen persistence and evolution during such relapsing infections are not well understood. Bacterial cells present in the primary inoculum and in late infections may differ greatly, as has been observed in chronic disease, or they may be genetically similar. To test these alternative models, we conducted whole-genome comparisons of clonal primary and relapse B. pseudomallei isolates recovered six months to six years apart from four adult Thai patients. We found differences within each of the four pairs, and some, including a 330 Kb deletion, affected substantial portions of the genome. Many of the changes were associated with increased antibiotic resistance. We also found evidence of positive selection for deleterious mutations in a TetR family transcriptional regulator from a set of 107 additional B. pseudomallei strains. As part of the study, we sequenced to base-pair accuracy the genome of B. pseudomallei strain 1026b, the model used for genetic studies of B. pseudomallei pathogenesis and antibiotic resistance. Our findings provide new insights into pathogen evolution during long-term infections and have important implications for the development of intervention strategies to combat recurrent melioidosis.

A comprehensive transposon mutant library of Francisella novicida, a bioweapon surrogate.

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is a category A select agent. We created a sequence-defined, near-saturation transposon mutant library of F. tularensis novicida, a subspecies that causes a tularemia-like disease in rodents. The library consists of 16,508 unique insertions, an average of >9 insertions per gene, which is a coverage nearly twice that of the greatest previously achieved for any bacterial species. Insertions were recovered in 84% (1,490) of the predicted genes. To achieve high coverage, it was necessary to construct transposons carrying an endogenous Francisella promoter to drive expression of antibiotic resistance. An analysis of genes lacking (or with few) insertions identified nearly 400 candidate essential genes, most of which are likely to be required for growth on rich medium and which represent potential therapeutic targets. To facilitate genome-scale screening using the mutant collection, we assembled a sublibrary made up of two purified mutants per gene. The library provides a resource for virtually complete identification of genes involved in virulence and other nonessential processes.