Arabinose and protocatechuate catabolism genes are important for growth of Rhizobium leguminosarum biovar viciae in the pea rhizosphere

Background and aims: To form nitrogen-fixing nodules on pea roots, Rhizobium leguminosarum biovar viciae must be competitive in the rhizosphere. Our aim was to identify genes important for rhizosphere fitness. Methods: Signature-tagged mutants were screened using microarrays to identify mutants reduced for growth in pea rhizospheres. Candidate mutants were assessed relative to controls for growth in minimal medium, growth in pea rhizospheres and for infection of peas in mixed inoculants. Mutated genes were identified by DNA sequencing and confirmed by transduction. Results: Of 5508 signature-tagged mutants, microarrays implicated 50 as having decreased rhizosphere fitness. Growth tests identified six mutants with rhizosphere-specific phenotypes. The mutation in one of the genes (araE) was in an arabinose catabolism operon and blocked growth on arabinose. The mutation in another gene (pcaM), encoding a predicted solute binding protein for protocatechuate and hydroxybenzoate uptake, decreased growth on protocatechuate. Both mutants were decreased for nodule infection competitiveness with mixed inoculants, but nodulated peas normally when inoculated alone. Other mutants with similar phenotypes had mutations predicted to affect secondary metabolism. Conclusions: Catabolism of arabinose and protocatechuate in the pea rhizosphere is important for competitiveness of R.l. viciae. Other genes predicted to be involved in secondary metabolism are also important

Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing

Global yields of potato and tomato crops are reduced owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, wild potato relatives are not and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow 1–3. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine R gene sequence capture (RenSeq4) with single-molecule real-time SMRT sequencing (SMRT RenSeq) to clone Rpi-amr3i . This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSEQ can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops

Growth Parameter Components of Adaptive Specificity during Experimental Evolution of the UVR-Inducible Mutator Pseudomonas cichorii 302959

BACKGROUND: Mutagenic DNA repair (MDR) transiently increases mutation rate through the activation of low-fidelity repair polymerases in response to specific, DNA-damaging environmental stress conditions such as ultraviolet radiation (UVR) exposure. These repair polymerases also confer UVR tolerance, intimately linking mutability and survival in bacteria that colone habitats subject to regular UVR exposure. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigate adaptive specificity in experimental lineages of the highly UVR-mutable epiphytic plant pathogen Pseudomonas cichorii 302959. Relative fitness measurements of isolates and population samples from replicate lineages indicated that adaptive improvements emerged early in all lineages of our evolution experiment and specific increases in relative fitness correlated with distinct improvements in doubling and lag times. Adaptive improvements gained under UVR and non-UVR conditions were acquired preferentially, and differentially contributed to relative fitness under varied growth conditions. CONCLUSIONS: These results support our earlier observations that MDR activation may contribute to gains in relative fitness without impeding normal patterns of adaptive specificity in P. cichorii 302959

Short clones or long clones? A simulation study on the use of paired reads in metagenomics

<p>Abstract</p> <p>Background</p> <p>Metagenomics is the study of environmental samples using sequencing. Rapid advances in sequencing technology are fueling a vast increase in the number and scope of metagenomics projects. Most metagenome sequencing projects so far have been based on Sanger or Roche-454 sequencing, as only these technologies provide long enough reads, while Illumina sequencing has not been considered suitable for metagenomic studies due to a short read length of only 35 bp. However, now that reads of length 75 bp can be sequenced in pairs, Illumina sequencing has become a viable option for metagenome studies.</p> <p>Results</p> <p>This paper addresses the problem of taxonomical analysis of paired reads. We describe a new feature of our metagenome analysis software MEGAN that allows one to process sequencing reads in pairs and makes assignments of such reads based on the combined bit scores of their matches to reference sequences. Using this new software in a simulation study, we investigate the use of Illumina paired-sequencing in taxonomical analysis and compare the performance of single reads, short clones and long clones. In addition, we also compare against simulated Roche-454 sequencing runs.</p> <p>Conclusion</p> <p>This work shows that paired reads perform better than single reads, as expected, but also, perhaps slightly less obviously, that long clones allow more specific assignments than short ones. A new version of the program MEGAN that explicitly takes paired reads into account is available from our website.</p

Bacterial adaptation is constrained in complex communities

© 2020, The Author(s). A major unresolved question is how bacteria living in complex communities respond to environmental changes. In communities, biotic interactions may either facilitate or constrain evolution depending on whether the interactions expand or contract the range of ecological opportunities. A fundamental challenge is to understand how the surrounding biotic community modifies evolutionary trajectories as species adapt to novel environmental conditions. Here we show that community context can dramatically alter evolutionary dynamics using a novel approach that ‘cages’ individual focal strains within complex communities. We find that evolution of focal bacterial strains depends on properties both of the focal strain and of the surrounding community. In particular, there is a stronger evolutionary response in low-diversity communities, and when the focal species have a larger genome and are initially poorly adapted. We see how community context affects resource usage and detect genetic changes involved in carbon metabolism and inter-specific interaction. The findings demonstrate that adaptation to new environmental conditions should be investigated in the context of interspecific interactions

BABAR: an R package to simplify the normalisation of common reference design microarray-based transcriptomic datasets

Background: The development of DNA microarrays has facilitated the generation of hundreds of thousands of transcriptomic datasets. The use of a common reference microarray design allows existing transcriptomic data to be readily compared and re-analysed in the light of new data, and the combination of this design with large datasets is ideal for 'systems' level analyses. One issue is that these datasets are typically collected over many years and may be heterogeneous in nature, containing different microarray file formats and gene array layouts, dye-swaps, and showing varying scales of log(2)- ratios of expression between microarrays. Excellent software exists for the normalisation and analysis of microarray data but many data have yet to be analysed as existing methods struggle with heterogeneous datasets; options include normalising microarrays on an individual or experimental group basis. Our solution was to develop the Batch Anti-Banana Algorithm in R (BABAR) algorithm and software package which uses cyclic loess to normalise across the complete dataset. We have already used BABAR to analyse the function of Salmonella genes involved in the process of infection of mammalian cells. Results: The only input required by BABAR is unprocessed GenePix or BlueFuse microarray data files. BABAR provides a combination of 'within' and 'between' microarray normalisation steps and diagnostic boxplots. When applied to a real heterogeneous dataset, BABAR normalised the dataset to produce a comparable scaling between the microarrays, with the microarray data in excellent agreement with RT-PCR analysis. When applied to a real non-heterogeneous dataset and a simulated dataset, BABAR's performance in identifying differentially expressed genes showed some benefits over standard techniques. Conclusions: BABAR is an easy-to-use software tool, simplifying the simultaneous normalisation of heterogeneous two-colour common reference design cDNA microarray-based transcriptomic datasets. We show BABAR transforms real and simulated datasets to allow for the correct interpretation of these data, and is the ideal tool to facilitate the identification of differentially expressed genes or network inference analysis from transcriptomic datasets

Read Length and Repeat Resolution: Exploring Prokaryote Genomes Using Next-Generation Sequencing Technologies

Background: There are a growing number of next-generation sequencing technologies. At present, the most cost-effective options also produce the shortest reads. However, even for prokaryotes, there is uncertainty concerning the utility of these technologies for the de novo assembly of complete genomes. This reflects an expectation that short reads will be unable to resolve small, but presumably abundant, repeats. Methodology/Principal Findings: Using a simple model of repeat assembly, we develop and test a technique that, for any read length, can estimate the occurrence of unresolvable repeats in a genome, and thus predict the number of gaps that would need to be closed to produce a complete sequence. We apply this technique to 818 prokaryote genome sequences. This provides a quantitative assessment of the relative performance of various lengths. Notably, unpaired reads of only 150nt can reconstruct approximately 50 % of the analysed genomes with fewer than 96 repeat-induced gaps. Nonetheless, there is considerable variation amongst prokaryotes. Some genomes can be assembled to near contiguity using very short reads while others require much longer reads. Conclusions: Given the diversity of prokaryote genomes, a sequencing strategy should be tailored to the organism unde

Defining Global Gene Expression Changes of the Hypothalamic-Pituitary-Gonadal Axis in Female sGnRH-Antisense Transgenic Common Carp (Cyprinus carpio)

BACKGROUND: The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. CONCLUSIONS/SIGNIFICANCE: This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of teleost fish

Pervasive Sign Epistasis between Conjugative Plasmids and Drug-Resistance Chromosomal Mutations

Multidrug-resistant bacteria arise mostly by the accumulation of plasmids and chromosomal mutations. Typically, these resistant determinants are costly to the bacterial cell. Yet, recently, it has been found that, in Escherichia coli bacterial cells, a mutation conferring resistance to an antibiotic can be advantageous to the bacterial cell if another antibiotic-resistance mutation is already present, a phenomenon called sign epistasis. Here we study the interaction between antibiotic-resistance chromosomal mutations and conjugative (i.e., self-transmissible) plasmids and find many cases of sign epistasis (40%)—including one of reciprocal sign epistasis where the strain carrying both resistance determinants is fitter than the two strains carrying only one of the determinants. This implies that the acquisition of an additional resistance plasmid or of a resistance mutation often increases the fitness of a bacterial strain already resistant to antibiotics. We further show that there is an overall antagonistic interaction between mutations and plasmids (52%). These results further complicate expectations of resistance reversal by interdiction of antibiotic use