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

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas fluorescens </it>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. <it>P. fluorescens </it>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.</p> <p>Results</p> <p>We used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for <it>P. fluorescens </it>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 <it>P. fluorescens </it>and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with <it>P. fluorescens </it>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.</p> <p>Conclusions</p> <p>The improved high-quality draft genome sequence of WH6 contributes towards resolving the <it>P. fluorescens </it>species, providing additional impetus for establishing two separate lineages in <it>P. fluorescens</it>. 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 <it>P. fluorescens </it>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.</p

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

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.Keywords: Regression, RNA-Seq, Overdispersion, Extra- Poisson variation, Negative binomial, Higher-order asymptotic

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

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

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Guanidine riboswitch-regulated efflux transporters protect bacteria against ionic liquid toxicity

Plant cell walls contain a renewable, nearly limitless supply of sugar that could be used to support microbial production of commodity chemicals and biofuels. Imidazolium ionic liquid (IIL) solvents are among the best reagents for gaining access to the sugars in this otherwise recalcitrant biomass. However, the sugars from IIL-treated biomass are inevitably contaminated with residual IILs that inhibit growth in bacteria and yeast, blocking biochemical production by these organisms. IIL toxicity is, therefore, a critical roadblock in many industrial biosynthetic pathways. Although several IIL-tolerant (IIL) bacterial and yeast isolates have been identified in nature, few genetic mechanisms have been identified. In this study, we identified two IIL Bacillus isolates as well as a spontaneous IIL Escherichia coli lab strain that are tolerant to high levels of two widely used IILs. We demonstrate that all three IIL strains contain one or more pumps of the small multidrug resistance (SMR) family, and two of these strains contain mutations that affect an adjacent regulatory guanidine riboswitch. Furthermore, we show that the regulation of E. coli sugE by the guanidine II riboswitch can be exploited to promote IIL tolerance by the simple addition of guanidine to the medium. Our results demonstrate the critical role that transporter genes play in IIL tolerance in their native bacterial hosts. The study presented here is another step in engineering IIL tolerance into industrial strains toward overcoming this key gap in biofuels and industrial biochemical production processes

Do Microcystis laboratory cultures hold clues to bacterial microcystin degradation?

Microcystins do not accumulate in freshwater ecosystems, and their degradation rates in freshwater suggest that both biotic and abiotic factors play critical roles in their removal. Most Microcystis aeruginosa laboratory cultures harbor a microbiome made up of heterotrophic bacteria that use Microcystis-produced organic matter for growth. We analyzed the microbiome of two M. aeruginosa cultures from Lake Erie with metagenomic sequencing, and found that some of their metagenome-assembled genomes included known genes for microcystin degradation. We tested whether these mixed communities can degrade microcystins by incubating them with Microcystis lysate (freeze-thawed cells) and monitoring dissolved microcystins by liquid chromatography mass spectrometry (LC/MS). We also tested whether these bacterial populations could incorporate carbon and nitrogen from microcystin-LR (MC-LR), the most common and most toxic of the microcystins, as well as bacteria from non-toxic Microcystis culture as a control. To accomplish this, we first labeled a M. aeruginosa culture with 13C and 15N stable isotopes, purified extracted MC-LR from the cell pellets, and added this labeled substrate back to Microcystis-bacteria co-cultures. Using nanoscale imaging mass spectrometry (NanoSIMS), we quantified the net incorporation of 15N and 13C into heterotrophic bacteria as well as the Microcystis cells to identify the fate of MC-LR C and N in this microbial ecosystem

New insights on organic nitrogen assimilation in Microcystis phycosphere and impacts on microcystin production

Microcystis is one of the most globally abundant bloom-forming cyanobacteria, comprising species that produce nitrogen-rich hepatotoxic compounds known as microcystins (MCs). Although dissolved organic nitrogen can exceed 50% of the nitrogen pool in aquatic ecosystems, organic nitrogen compounds are often overlooked in terms of their impact on community dynamics and Microcystis bloom development. Further, cyanobacteria are constantly interacting with other microorganisms in their surrounding environment, but nitrogen cycling in the phycosphere is poorly understood. In the present study, we monitored the growth and MC production of several Microcystis strains on various organic nitrogen sources including amino acids and proteins, investigated the impacts of organic nitrogen on Microcystis microbiome via amplicon and metagenomic sequencing, and traced the nitrogen assimilation within the phycosphere at the single-cell level by measuring isotopic tracer incorporation via secondary ion mass spectrometer imaging (NanoSIMS). We demonstrate that 1) organic nitrogen species shape the microbiome community structure in the Microcystisphycosphere, and 2) competition for and/or transport of nitrogen between heterotrophic bacteria and cyanobacteria potentially play important roles for cyanobacterial succession especially under inorganic nitrogen scarcity. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA2734

Mutualistic Co-evolution of Type III Effector Genes in <em>Sinorhizobium fredii</em> and <em>Bradyrhizobium japonicum</em>

<div><p>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, <i>Sinorhizobium fredii</i> and <i>Bradyrhizobium japonicum</i>. 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.</p> </div