Jeffrey A. Kimbrel

Recombineering and stable integration of the Pseudomonas syringae pv. syringae 61 hrp/hrc cluster into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1.

Many Gram-negative bacteria use a type III secretion system (T3SS) to establish associations with their hosts. The T3SS is a conduit for direct injection of type-III effector proteins into host cells, where they manipulate the host for the benefit of the infecting bacterium. For plant-associated pathogens, the variations in number and amino acid sequences of type-III effectors, as well as their functional redundancy, make studying type-III effectors challenging. To mitigate this challenge, we developed a stable delivery system for individual or defined sets of type-III effectors into plant cells. We used recombineering and Tn5-mediated transposition to clone and stably integrate, respectively, the complete hrp/hrc region from Pseudomonas syringae pv. syringae 61 into the genome of the soil bacterium Pseudomonas fluorescens Pf0-1. We describe our development of Effector-to-Host Analyzer (EtHAn), and demonstrate its utility for studying effectors for their in planta functions.

GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts.

An improved, high-quality draft genome sequence of the Germination-Arrest Factor-producing Pseudomonas fluorescens WH6

BackgroundPseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. P. fluorescens isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species.ResultsWe used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for P. fluorescens WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to P. fluorescens and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with P. fluorescens SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system.ConclusionsThe improved high-quality draft genome sequence of WH6 contributes towards resolving the P. fluorescens species, providing additional impetus for establishing two separate lineages in P. fluorescens. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of P. fluorescens reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.

The membrane‐associated monooxygenase in the butane‐oxidizing Gram‐positive bacterium Nocardioides sp. strain CF8 is a novel member of the AMO/PMO family

The Gram-positive bacterium Nocardioides sp. strain CF8 uses a membrane-associated monooxygenase (pBMO) to grow on butane. The nucleotide sequences of the genes encoding this novel monooxygenase were revealed through analysis of a de novo assembled draft genome sequence determined by high-throughput sequencing of the whole genome. The pBMO genes were in a similar arrangement to the genes for ammonia monooxygenase (AMO) from the ammonia-oxidizing bacteria and for particulate methane monooxygenase (pMMO) from the methane-oxidizing bacteria. The pBMO genes likely constitute an operon in the order bmoC, bmoA and bmoB. The nucleotide sequence was less than 50% similar to the genes for AMO and pMMO. The operon for pBMO was confirmed to be a single copy in the genome by Southern and computational analyses. In an incubation on butane the increase of transcriptional activity of the pBmoA gene was congruent with the increase of pBMO activity and suggested correspondence between gene expression and the utilization of butane. Phylogenetic comparison revealed distant but significant similarity of all three pBMO subunits to homologous members of the AMO/pMMO family and indicated that the pBMO represents a deeply branching third lineage of this group of particulate monooxygenases. No other bmoCAB-like genes were found to cluster with pBMO lineage in phylogenetic analysis by database searches including genomic and metagenomic sequence databases. pBMO is the first example of the AMO/pMMO-like monooxygenase from Gram-positive bacteria showing similarities to proteobacterial pMMO and AMO sequences.

Mutualistic co-evolution of type III effector genes in Sinorhizobium fredii and Bradyrhizobium japonicum.

Two diametric paradigms have been proposed to model the molecular co-evolution of microbial mutualists and their eukaryotic hosts. In one, mutualist and host exhibit an antagonistic arms race and each partner evolves rapidly to maximize their own fitness from the interaction at potential expense of the other. In the opposing model, conflicts between mutualist and host are largely resolved and the interaction is characterized by evolutionary stasis. We tested these opposing frameworks in two lineages of mutualistic rhizobia, Sinorhizobium fredii and Bradyrhizobium japonicum. To examine genes demonstrably important for host-interactions we coupled the mining of genome sequences to a comprehensive functional screen for type III effector genes, which are necessary for many Gram-negative pathogens to infect their hosts. We demonstrate that the rhizobial type III effector genes exhibit a surprisingly high degree of conservation in content and sequence that is in contrast to those of a well characterized plant pathogenic species. This type III effector gene conservation is particularly striking in the context of the relatively high genome-wide diversity of rhizobia. The evolution of rhizobial type III effectors is inconsistent with the molecular arms race paradigm. Instead, our results reveal that these loci are relatively static in rhizobial lineages and suggest that fitness conflicts between rhizobia mutualists and their host plants have been largely resolved.

Sequencing and De Novo Transcriptome Assembly of Brachypodium sylvaticum (Poaceae)

Premise of the study: We report the de novo assembly and characterization of the transcriptomes of Brachypodium sylvaticum (slender false-brome) accessions from native populations of Spain and Greece, and an invasive population west of Corvallis, Oregon, USA. Methods and Results: More than 350 million sequence reads from the mRNA libraries prepared from three B. sylvaticum genotypes were assembled into 120,091 (Corvallis), 104,950 (Spain), and 177,682 (Greece) transcript contigs. In comparison with the B. distachyon Bd21 reference genome and GenBank protein sequences, we estimate >90% exome coverage for B. sylvaticum. The transcripts were assigned Gene Ontology and InterPro annotations. Brachypodium sylvaticum sequence reads aligned against the Bd21 genome revealed 394,654 single-nucleotide polymorphisms (SNPs) and >20,000 simple sequence repeat (SSR) DNA sites. Conclusions: To our knowledge, this is the first report of transcriptome sequencing of invasive plant species with a closely related sequenced reference genome. The sequences and identified SNP variant and SSR sites will provide tools for developing novel genetic markers for use in genotyping and characterization of invasive behavior of B. sylvaticum.

Genome sequencing and comparative analysis of the carrot bacterial blight pathogen, Xanthomonas hortorum pv. carotae M081, for insights into pathogenicity and applications in molecular diagnostics

Xanthomonas hortorum pv. carotae (Xhc) is an economically important pathogen of carrots. Its ability to epiphytically colonize foliar surfaces and infect seeds can result in bacterial blight of carrots when grown in warm and humid regions. We used high-throughput sequencing to determine the genome sequence of isolate M081 of Xhc. The short reads were de novo assembled and the resulting contigs were ordered using a syntenic reference genome sequence from X. campestris pv. campestris ATCC 33913. The improved, high-quality draft genome sequence of Xhc M081 is the first for its species. Despite its distance from other sequenced xanthomonads, Xhc M081 still shared a large inventory of orthologous genes, including many clusters of virulence genes common to other foliar pathogenic species of Xanthomonas. We also mined the genome sequence and identified at least 21 candidate type III effector genes. Two were members of the avrBs2 and xopQ families that demonstrably elicit effector-triggered immunity. We showed that expression in planta of these two type III effectors from Xhc M081 was sufficient to elicit resistance gene-mediated hypersensitive responses in heterologous plants, indicating a possibility for resistance gene-mediated control of Xhc. Finally, we identified regions unique to the Xhc M081 genome sequence, and demonstrated their potential in the design of molecular diagnostics for this pathogen.

Higher order asymptotics for negative binomial regression inferences from RNA-sequencing data

Abstract RNA sequencing (RNA-Seq) is the current method of choice for characterizing transcriptomes and quantifying gene expression changes. This next generation sequencing-based method provides unprecedented depth and resolution. The negative binomial (NB) probability distribution has been shown to be a useful model for frequencies of mapped RNA-Seq reads and consequently provides a basis for statistical analysis of gene expression. Negative binomial exact tests are available for two-group comparisons but do not extend to negative binomial regression analysis, which is important for examining gene expression as a function of explanatory variables and for adjusted group comparisons accounting for other factors. We address the adequacy of available large-sample tests for the small sample sizes typically available from RNA-Seq studies and consider a higher-order asymptotic (HOA) adjustment to likelihood ratio tests. We demonstrate that 1) the HOA-adjusted likelihood ratio test is practically indistinguishable from the exact test in situations where the exact test is available, 2) the type I error of the HOA test matches the nominal specification in regression settings we examined via simulation, and 3) the power of the likelihood ratio test does not appear to be affected by the HOA adjustment. This work helps clarify the accuracy of the unadjusted likelihood ratio test and the degree of improvement available with the HOA adjustment. Furthermore, the HOA test may be preferable even when the exact test is available because it does not require ad hoc library size adjustments.

RNA-Seq for Plant Pathogenic Bacteria

The throughput and single-base resolution of RNA-Sequencing (RNA-Seq) have contributed to a dramatic change in transcriptomic-based inquiries and resulted in many new insights into the complexities of bacterial transcriptomes. RNA-Seq could contribute to similar advances in our understanding of plant pathogenic bacteria but it is still a technology under development with limitations and unknowns that need to be considered. Here, we review some new developments for RNA-Seq and highlight recent findings for host-associated bacteria. We also discuss the technical and statistical challenges in the practical application of RNA-Seq for studying bacterial transcriptomes and describe some of the currently available solutions.

The Draft Genome Sequence of Nocardioides sp. Strain CF8 Reveals the Scope of Its Metabolic Capabilities.

ABSTRACT Nocardioides sp. strain CF8 was isolated from a soil sample collected at the Hanford Department of Energy site, Richland, WA. The strain was identified in microcosms based on its ability to grow on butane and has been characterized for its potential applications in the biodegradation of halogenated hydrocarbons. Here, the draft genome sequence is reported.