Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts.

The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a monospecific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that likely contributes to establish and control the gut microbiota.

Genomics in the long-read sequencing era.

Long-read sequencing (LRS) technologies have provided extremely powerful tools to explore genomes. While in the early years these methods suffered technical limitations, they have recently made significant progress in terms of read length, throughput, and accuracy and bioinformatics tools have strongly improved. Here, we aim to review the current status of LRS technologies, the development of novel methods, and the impact on genomics research. We will explore the most impactful recent findings made possible by these technologies focusing on high-resolution sequencing of genomes and transcriptomes and the direct detection of DNA and RNA modifications. We will also discuss how LRS methods promise a more comprehensive understanding of human genetic variation, transcriptomics, and epigenetics for the coming years.

Pervasive transcription enhances the accessibility of H-NS-silenced promoters and generates bistability in Salmonella virulence gene expression.

In Escherichia coli and Salmonella, many genes silenced by the nucleoid structuring protein H-NS are activated upon inhibiting Rho-dependent transcription termination. This response is poorly understood and difficult to reconcile with the view that H-NS acts mainly by blocking transcription initiation. Here we have analyzed the basis for the up-regulation of H-NS-silenced Salmonella pathogenicity island 1 (SPI-1) in cells depleted of Rho-cofactor NusG. Evidence from genetic experiments, semiquantitative 5' rapid amplification of complementary DNA ends sequencing (5' RACE-Seq), and chromatin immunoprecipitation sequencing (ChIP-Seq) shows that transcription originating from spurious antisense promoters, when not stopped by Rho, elongates into a H-NS-bound regulatory region of SPI-1, displacing H-NS and rendering the DNA accessible to the master regulator HilD. In turn, HilD's ability to activate its own transcription triggers a positive feedback loop that results in transcriptional activation of the entire SPI-1. Significantly, single-cell analyses revealed that this mechanism is largely responsible for the coexistence of two subpopulations of cells that either express or do not express SPI-1 genes. We propose that cell-to-cell differences produced by stochastic spurious transcription, combined with feedback loops that perpetuate the activated state, can generate bimodal gene expression patterns in bacterial populations.

Contrasting Gene Decay in Subterranean Vertebrates: Insights from Cavefishes and Fossorial Mammals.

Evolution sometimes proceeds by loss, especially when structures and genes become dispensable after an environmental shift relaxes functional constraints. Subterranean vertebrates are outstanding models to analyze this process, and gene decay can serve as a readout. We sought to understand some general principles on the extent and tempo of the decay of genes involved in vision, circadian clock, and pigmentation in cavefishes. The analysis of the genomes of two Cuban species belonging to the genus Lucifuga provided evidence for the largest loss of eye-specific genes and nonvisual opsin genes reported so far in cavefishes. Comparisons with a recently evolved cave population of Astyanax mexicanus and three species belonging to the Chinese tetraploid genus Sinocyclocheilus revealed the combined effects of the level of eye regression, time, and genome ploidy on eye-specific gene pseudogenization. The limited extent of gene decay in all these cavefishes and the very small number of loss-of-function mutations per pseudogene suggest that their eye degeneration may not be very ancient, ranging from early to late Pleistocene. This is in sharp contrast with the identification of several vision genes carrying many loss-of-function mutations in ancient fossorial mammals, further suggesting that blind fishes cannot thrive more than a few million years in cave ecosystems.

Agrobacterium tumefaciens fitness genes involved in the colonization of plant tumors and roots.

Agrobacterium tumefaciens colonizes the galls (plant tumors) it causes, and the roots of host and nonhost plants. Transposon-sequencing (Tn-Seq) was used to discover A.tumefaciens genes involved in reproductive success (fitness genes) on Solanum lycopersicum and Populus trichocarpa tumors and S.lycopersicum and Zea mays roots. The identified fitness genes represent 3-8% of A. tumefaciens genes and contribute to carbon and nitrogen metabolism, synthesis and repair of DNA, RNA and proteins and envelope-associated functions. Competition assays between 12 knockout mutants and wild-type confirmed the involvement of 10 genes (trpB, hisH, metH, cobN, ntrB, trxA, nrdJ, kamA, exoQ, wbbL) in A.tumefaciens fitness under both tumor and root conditions. The remaining two genes (fecA, noxA) were important in tumors only. None of these mutants was nonpathogenic, but four (hisH, trpB, exoQ, ntrB) exhibited impaired virulence. Finally, we used this knowledge to search for chemical and biocontrol treatments that target some of the identified fitness pathways and report reduced tumorigenesis and impaired establishment of A.tumefaciens on tomato roots using tannic acid or Pseudomonas protegens, which affect iron assimilation. This work revealed A.tumefaciens pathways that contribute to its competitive survival in plants and highlights a strategy to identify plant protection approaches against this pathogen.

The Third Revolution in Sequencing Technology.

Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives.

The biotroph Agrobacterium tumefaciens thrives in tumors by exploiting a wide spectrum of plant host metabolites.

Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics, and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host plant. Metabolomics of healthy stems and plant tumors revealed the common (e.g. sucrose, glutamate) and enriched (e.g. opines, γ-aminobutyric acid (GABA), γ-hydroxybutyric acid (GHB), pyruvate) metabolites that A. tumefaciens could use as nutrients. Tn-seq and transcriptomics pinpointed the genes that are crucial and/or upregulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, TCA cycle, and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA, and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge), and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in plant tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in a plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and Tn-seq to decipher the lifestyle of a plant pathogen.

Systematic comparison of small RNA library preparation protocols for next-generation sequencing.

BACKGROUND: Next-generation sequencing technologies have revolutionized the study of small RNAs (sRNAs) on a genome-wide scale. However, classical sRNA library preparation methods introduce serious bias, mainly during adapter ligation steps. Several types of sRNA including plant microRNAs (miRNA), piwi-interacting RNAs (piRNA) in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants contain a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This inhibits 3' adapter ligation and makes library preparation particularly challenging. To reduce bias, the NEBNext kit (New England Biolabs) uses polyethylene glycol (PEG), the NEXTflex V2 kit (BIOO Scientific) uses both randomised adapters and PEG, and the novel SMARTer (Clontech) and CATS (Diagenode) kits avoid ligation altogether. Here we compared these methods with Illumina's classical TruSeq protocol regarding the detection of normal and 2' OMe RNAs. In addition, we modified the TruSeq and NEXTflex protocols to identify conditions that improve performance. RESULTS: Among the five kits tested with their respective standard protocols, the SMARTer and CATS kits had the lowest levels of bias but also had a strong formation of side products, and as a result performed relatively poorly with biological samples; NEXTflex detected the largest numbers of different miRNAs. The use of a novel type of randomised adapters called MidRand-Like (MRL) adapters and PEG improved the detection of 2' OMe RNAs both in the TruSeq as well as in the NEXTflex protocol. CONCLUSIONS: While it is commonly accepted that biases in sRNA library preparation protocols are mainly due to adapter ligation steps, the ligation-free protocols were not the best performing methods. Our modified versions of the TruSeq and NEXTflex protocols provide an improved tool for the study of 2' OMe RNAs.

Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.

Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions.

Suppression of Dwarf and irregular xylem Phenotypes Generates Low-Acetylated Biomass Lines in Arabidopsis.

eskimo1-5 (esk1-5) is a dwarf Arabidopsis (Arabidopsis thaliana) mutant that has a constitutive drought syndrome and collapsed xylem vessels, along with low acetylation levels in xylan and mannan. ESK1 has xylan O-acetyltransferase activity in vitro. We used a suppressor strategy on esk1-5 to screen for variants with wild-type growth and low acetylation levels, a favorable combination for ethanol production. We found a recessive mutation in the KAKTUS (KAK) gene that suppressed dwarfism and the collapsed xylem character, the cause of decreased hydraulic conductivity in the esk1-5 mutant. Backcrosses between esk1-5 and two independent knockout kak mutants confirmed suppression of the esk1-5 effect. kak single mutants showed larger stem diameters than the wild type. The KAK promoter fused with a reporter gene showed activity in the vascular cambium, phloem, and primary xylem in the stem and hypocotyl. However, suppression of the collapsed xylem phenotype in esk1 kak double mutants was not associated with the recovery of cell wall O-acetylation or any major cell wall modifications. Therefore, our results indicate that, in addition to its described activity as a repressor of endoreduplication, KAK may play a role in vascular development. Furthermore, orthologous esk1 kak double mutants may hold promise for ethanol production in crop plants.

CIRCUS: a package for Circos display of structural genome variations from paired-end and mate-pair sequencing data.

BACKGROUND: Detection of large genomic rearrangements, such as large indels, duplications or translocations is now commonly achieved by next generation sequencing (NGS) approaches. Recently, several tools have been developed to analyze NGS data but the resulting files are difficult to interpret without an additional visualization step. Circos (Genome Res, 19:1639-1645, 2009), a Perl script, is a powerful visualization software that requires setting up numerous configuration files with a large number of parameters to handle. R packages like RCircos (BMC Bioinformatics, 14:244, 2013) or ggbio (Genome Biol, 13:R77, 2012) provide functions to display genomic data as circular Circos-like plots. However, these tools are very general and lack the functions needed to filter, format and adjust specific input genomic data. RESULTS: We implemented an R package called CIRCUS to analyze genomic structural variations. It generates both data and configuration files necessary for Circos, to produce graphs. Only few R pre-requisites are necessary. Options are available to deal with heterogeneous data, various chromosome numbers and multi-scale analysis. CONCLUSION: CIRCUS allows fast and versatile analysis of genomic structural variants with Circos plots for users with limited coding skills.

Assemblathon 1: a competitive assessment of de novo short read assembly methods.

Low-cost short read sequencing technology has revolutionized genomics, though it is only just becoming practical for the high-quality de novo assembly of a novel large genome. We describe the Assemblathon 1 competition, which aimed to comprehensively assess the state of the art in de novo assembly methods when applied to current sequencing technologies. In a collaborative effort, teams were asked to assemble a simulated Illumina HiSeq data set of an unknown, simulated diploid genome. A total of 41 assemblies from 17 different groups were received. Novel haplotype aware assessments of coverage, contiguity, structure, base calling, and copy number were made. We establish that within this benchmark: (1) It is possible to assemble the genome to a high level of coverage and accuracy, and that (2) large differences exist between the assemblies, suggesting room for further improvements in current methods. The simulated benchmark, including the correct answer, the assemblies, and the code that was used to evaluate the assemblies is now public and freely available from http://www.assemblathon.org/.

The first complete chloroplast genome of the Genistoid legume Lupinus luteus: evidence for a novel major lineage-specific rearrangement and new insights regarding plastome evolution in the legume family.

BACKGROUND AND AIMS: To date chloroplast genomes are available only for members of the non-protein amino acid-accumulating clade (NPAAA) Papilionoid lineages in the legume family (i.e. Millettioids, Robinoids and the 'inverted repeat-lacking clade', IRLC). It is thus very important to sequence plastomes from other lineages in order to better understand the unusual evolution observed in this model flowering plant family. To this end, the plastome of a lupine species, Lupinus luteus, was sequenced to represent the Genistoid lineage, a noteworthy but poorly studied legume group. METHODS: The plastome of L. luteus was reconstructed using Roche-454 and Illumina next-generation sequencing. Its structure, repetitive sequences, gene content and sequence divergence were compared with those of other Fabaceae plastomes. PCR screening and sequencing were performed in other allied legumes in order to determine the origin of a large inversion identified in L. luteus. KEY RESULTS: The first sequenced Genistoid plastome (L. luteus: 155 894 bp) resulted in the discovery of a 36-kb inversion, embedded within the already known 50-kb inversion in the large single-copy (LSC) region of the Papilionoideae. This inversion occurs at the base or soon after the Genistoid emergence, and most probably resulted from a flip-flop recombination between identical 29-bp inverted repeats within two trnS genes. Comparative analyses of the chloroplast gene content of L. luteus vs. Fabaceae and extra-Fabales plastomes revealed the loss of the plastid rpl22 gene, and its functional relocation to the nucleus was verified using lupine transcriptomic data. An investigation into the evolutionary rate of coding and non-coding sequences among legume plastomes resulted in the identification of remarkably variable regions. CONCLUSIONS: This study resulted in the discovery of a novel, major 36-kb inversion, specific to the Genistoids. Chloroplast mutational hotspots were also identified, which contain novel and potentially informative regions for molecular evolutionary studies at various taxonomic levels in the legumes. Taken together, the results provide new insights into the evolutionary landscape of the legume plastome.

Transposon sequencing reveals the essential gene set and genes enabling gut symbiosis in the insect symbiont Caballeronia insecticola.

Caballeronia insecticola is a bacterium belonging to the Burkholderia genus sensu lato, which is able to colonize multiple environments like soils and the gut of the bean bug Riptortus pedestris. We constructed a saturated Himar1 mariner transposon library and revealed by transposon-sequencing that 498 protein-coding genes constitute the essential genome of Caballeronia insecticola for growth in free-living conditions. By comparing essential gene sets of Caballeronia insecticola and seven related Burkholderia s.l. strains, only 120 common genes were identified, indicating that a large part of the essential genome is strain-specific. In order to reproduce specific nutritional conditions that are present in the gut of Riptortus pedestris, we grew the mutant library in minimal media supplemented with candidate gut nutrients and identified several condition-dependent fitness-defect genes by transposon-sequencing. To validate the robustness of the approach, insertion mutants in six fitness genes were constructed and their growth deficiency in media supplemented with the corresponding nutrient was confirmed. The mutants were further tested for their efficiency in Riptortus pedestris gut colonization, confirming that gluconeogenic carbon sources, taurine and inositol, are nutrients consumed by the symbiont in the gut. Thus, our study provides insights about specific contributions provided by the insect host to the bacterial symbiont.

Expression of Dystrophin Dp71 Splice Variants Is Temporally Regulated During Rodent Brain Development.

Dystrophin Dp71 is the major product of the Duchenne muscular dystrophy (DMD) gene in the brain, and its loss in DMD patients and mouse models leads to cognitive impairments. Dp71 is expressed as a range of proteins generated by alternative splicing of exons 71 to 74 and 78, classified in the main Dp71d and Dp71f groups that contain specific C-terminal ends. However, it is unknown whether each isoform has a specific role in distinct cell types, brain regions, and/or stages of brain development. In the present study, we characterized the expression of Dp71 isoforms during fetal (E10.5, E15.5) and postnatal (P1, P7, P14, P21 and P60) mouse and rat brain development. We finely quantified the expression of several Dp71 transcripts by RT-PCR and cloning assays in samples from whole-brain and distinct brain structures. The following Dp71 transcripts were detected: Dp71d, Dp71d∆71, Dp71d∆74, Dp71d∆71,74, Dp71d∆71-74, Dp71f, Dp71f∆71, Dp71f∆74, Dp71f∆71,74, and Dp71fΔ71-74. We found that the Dp71f isoform is the main transcript expressed at E10.5 (> 80%), while its expression is then progressively reduced and replaced by the expression of isoforms of the Dp71d group from E15.5 to postnatal and adult ages. This major finding was confirmed by third-generation nanopore sequencing. In addition, we found that the level of expression of specific Dp71 isoforms varies as a function of postnatal stages and brain structure. Our results suggest that Dp71 isoforms have different and complementary roles during embryonic and postnatal brain development, likely taking part in a variety of maturation processes in distinct cell types.

Construction of a Transposon Mutant Library in the Pathogen Agrobacterium tumefaciens C58 and Identification of Genes Involved in Gall Niche Exploitation and Colonization.

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease on a wide range of host species by transferring and integrating a part of its own DNA (T-DNA) into the plant genome. The genes responsible of the above-mentioned processes are well characterized. However, a large number of the mechanisms involved in exploitation and colonization of the galls (also named plant tumors) remain unknown. Due to recent development of "transposon-sequencing" (Tn-Seq) techniques, a high-throughput screening and identification of the different genes involved in such mechanisms is now possible. In this chapter, we describe the detailed methodology used to construct a transposon library in A. tumefaciens and to conduct a Tn-Seq approach to discover genes involved in plant tumor exploitation and colonization.

Dissimilar gene repertoires of Dickeya solani involved in the colonization of lesions and roots of Solanum tuberosum.

Dickeya and Pectobacterium species are necrotrophic pathogens that macerate stems (blackleg disease) and tubers (soft rot disease) of Solanum tuberosum. They proliferate by exploiting plant cell remains. They also colonize roots, even if no symptoms are observed. The genes involved in pre-symptomatic root colonization are poorly understood. Here, transposon-sequencing (Tn-seq) analysis of Dickeya solani living in macerated tissues revealed 126 genes important for competitive colonization of tuber lesions and 207 for stem lesions, including 96 genes common to both conditions. Common genes included acr genes involved in the detoxification of plant defense phytoalexins and kduD, kduI, eda (=kdgA), gudD, garK, garL, and garR genes involved in the assimilation of pectin and galactarate. In root colonization, Tn-seq highlighted 83 genes, all different from those in stem and tuber lesion conditions. They encode the exploitation of organic and mineral nutrients (dpp, ddp, dctA, and pst) including glucuronate (kdgK and yeiQ) and synthesis of metabolites: cellulose (celY and bcs), aryl polyene (ape), and oocydin (ooc). We constructed in-frame deletion mutants of bcsA, ddpA, apeH, and pstA genes. All mutants were virulent in stem infection assays, but they were impaired in the competitive colonization of roots. In addition, the ΔpstA mutant was impaired in its capacity to colonize progeny tubers. Overall, this work distinguished two metabolic networks supporting either an oligotrophic lifestyle on roots or a copiotrophic lifestyle in lesions. This work revealed novel traits and pathways important for understanding how the D. solani pathogen efficiently survives on roots, persists in the environment, and colonizes progeny tubers.

Construction and characterization of a saturated Tn-seq library of Salmonella Typhimurium ATCC 14028.

Salmonella enterica is an important foodborne pathogen. Here, we present the construction and characterization of a high-density transposon sequencing library of the Salmonella Typhimurium ATCC 14028 strain. Essential, advantageous, and disadvantageous genes for growth in rich culture medium were identified on the chromosome and the pSLT plasmid.

Plesiomonas shigelloides, an Atypical Enterobacterales with a Vibrio-Related Secondary Chromosome.

About 10% of bacteria have a multichromosome genome with a primary replicon of bacterial origin, called the chromosome, and other replicons of plasmid origin, the chromids. Studies on multichromosome bacteria revealed potential points of coordination between the replication/segregation of chromids and the progression of the cell cycle. For example, replication of the chromid of Vibrionales (called Chr2) is initiated upon duplication of a sequence carried by the primary chromosome (called Chr1), in such a way that replication of both replicons is completed synchronously. Also, Chr2 uses the Chr1 as a scaffold for its partition in the daughter cells. How many of the features detected so far are required for the proper integration of a secondary chromosome in the cell cycle? How many more features remain to be discovered? We hypothesized that critical features for the integration of the replication/segregation of a given chromid within the cell cycle program would be conserved independently of the species in which the chromid has settled. Hence, we searched for a chromid related to that found in Vibrionales outside of this order. We identified one in Plesiomonas shigelloides, an aquatic and pathogenic enterobacterium that diverged early within the clade of Enterobacterales. Our results suggest that the chromids present in P. shigelloides and Vibrionales derive from a common ancestor. We initiated in silico genomic and proteomic comparative analyses of P. shigelloides, Vibrionales, and Enterobacterales that enabled us to establish a list of features likely involved in the maintenance of the chromid within the host cell cycle.

Genome Sequences of 11 Conspecific Streptomyces sp. Strains.

The genomes of 11 conspecific Streptomyces strains, i.e., from the same species and inhabiting the same ecological niche, were sequenced and assembled. This data set offers an ideal framework to assess the genome evolution of Streptomyces species in their ecological context.