Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.

Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.

Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici.

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.

Rapid, specific, simple, in-field detection of Xanthomonas campestris pathovar musacearum by loop-mediated isothermal amplification.

AIMS: To develop and evaluate a loop-mediated isothermal amplification (LAMP) assay for Xanthomonas campestris pathovar musacearum (Xcm), the causal agent of banana Xanthomonas wilt, a major disease of banana in Africa. METHODS AND RESULTS: LAMP primers were designed to the general secretion pathway protein D gene and tested against 17 isolates of Xcm encompassing the known genetic and geographic diversity of the bacterium and all isolates were detected. Seventeen other Xanthomonas isolates, including closely related Xanthomonas vasicola, other bacterial pathogens/endophytes of Musa and two healthy Musa varieties gave negative results with the LAMP assay. The assay showed good sensitivity, detecting as little as 51 fg of Xcm DNA, a greater level of sensitivity than that of an Xcm PCR assay. Amplification with the LAMP assay was very rapid, typically within 9 min from bacterial cultures. Symptomatic field samples of Musa from Uganda were tested and all produced amplification in less than 13 min. CONCLUSIONS: The LAMP assay provides rapid, sensitive detection of the pathogen that is ideally suited for deployment in laboratories with basic facilities and in-field situations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first LAMP assay for Xcm which provides a significant improvement compared to existing diagnostics.

Publisher Correction: Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom.

The PDF version of this Article was updated shortly after publication following an error which resulted in the Φ symbol being omitted from the left hand side of equation 8. The HTML version was correct from the time of publication.

Environmental fluctuations accelerate molecular evolution of thermal tolerance in a marine diatom.

Diatoms contribute roughly 20% of global primary production, but the factors determining their ability to adapt to global warming are unknown. Here we quantify the capacity for adaptation to warming in the marine diatom Thalassiosira pseudonana. We find that evolutionary rescue under severe (32 °C) warming is slow, but adaptation to more realistic scenarios where temperature increases are moderate (26 °C) or fluctuate between benign and severe conditions is rapid and linked to phenotypic changes in metabolic traits and elemental composition. Whole-genome re-sequencing identifies genetic divergence among populations selected in the different warming regimes and between the evolved and ancestral lineages. Consistent with the phenotypic changes, the most rapidly evolving genes are associated with transcriptional regulation, cellular responses to oxidative stress and redox homeostasis. These results demonstrate that the evolution of thermal tolerance in marine diatoms can be rapid, particularly in fluctuating environments, and is underpinned by major genomic and phenotypic change.

Genome sequences of six Phytophthora species associated with forests in New Zealand.

In New Zealand there has been a long association of Phytophthora diseases in forests, nurseries, remnant plantings and horticultural crops. However, new Phytophthora diseases of trees have recently emerged. Genome sequencing has been performed for 12 Phytophthora isolates, from six species: Phytophthora pluvialis, Phytophthora kernoviae, Phytophthora cinnamomi, Phytophthora agathidicida, Phytophthora multivora and Phytophthora taxon Totara. These sequences will enable comparative analyses to identify potential virulence strategies and ultimately facilitate better control strategies. This Whole Genome Shotgun data have been deposited in DDBJ/ENA/GenBank under the accession numbers LGTT00000000, LGTU00000000, JPWV00000000, JPWU00000000, LGSK00000000, LGSJ00000000, LGTR00000000, LGTS00000000, LGSM00000000, LGSL00000000, LGSO00000000, and LGSN00000000.

Transcriptomic analyses support the similarity of gene expression between brain and testis in human as well as mouse.

We previously revealed similarity in gene expression patterns between human brain and testis, based on digital differential display analyses of 760 human Unigenes. In the present work, we reanalyzed the gene expression data in many tissues of human and mouse for a large number of genes almost covering the respective whole genomes. The results indicated that both in human and in mouse, the gene expression profiles exhibited by brain, cerebellum and testis are most similar to each other compared with other tissues.

Development of a lateral flow device for in-field detection and evaluation of PCR-based diagnostic methods for Xanthomonas campestris pv. musacearum, the causal agent of banana xanthomonas wilt.

Xanthomonas campestris pv. musacearum (Xcm) is the causal agent of banana xanthomonas wilt, a major threat to banana production in eastern and central Africa. The pathogen is present in very high levels within infected plants and can be transmitted by a broad range of mechanisms; therefore early specific detection is vital for effective disease management. In this study, a polyclonal antibody (pAb) was developed and deployed in a lateral flow device (LFD) format to allow rapid in-field detection of Xcm. Published Xcm PCR assays were also independently assessed: only two assays gave specific amplification of Xcm, whilst others cross-reacted with non-target Xanthomonas species. Pure cultures of Xcm were used to immunize a rabbit, the IgG antibodies purified from the serum and the resulting polyclonal antibodies tested using ELISA and LFD. Testing against a wide range of bacterial species showed the pAb detected all strains of Xcm, representing isolates from seven countries and the known genetic diversity of Xcm. The pAb also detected the closely related Xanthomonas axonopodis pv. vasculorum (Xav), primarily a sugarcane pathogen. Detection was successful in both naturally and experimentally infected banana plants, and the LFD limit of detection was 105 cells mL-1. Whilst the pAb is not fully specific for Xcm, Xav has never been found in banana. Therefore the LFD can be used as a first-line screening tool to detect Xcm in the field. Testing by LFD requires no equipment, can be performed by non-scientists and is cost-effective. Therefore this LFD provides a vital tool to aid in the management and control of Xcm.

Assessing the performance of the Oxford Nanopore Technologies MinION.

The Oxford Nanopore Technologies (ONT) MinION is a new sequencing technology that potentially offers read lengths of tens of kilobases (kb) limited only by the length of DNA molecules presented to it. The device has a low capital cost, is by far the most portable DNA sequencer available, and can produce data in real-time. It has numerous prospective applications including improving genome sequence assemblies and resolution of repeat-rich regions. Before such a technology is widely adopted, it is important to assess its performance and limitations in respect of throughput and accuracy. In this study we assessed the performance of the MinION by re-sequencing three bacterial genomes, with very different nucleotide compositions ranging from 28.6% to 70.7%; the high G + C strain was underrepresented in the sequencing reads. We estimate the error rate of the MinION (after base calling) to be 38.2%. Mean and median read lengths were 2 kb and 1 kb respectively, while the longest single read was 98 kb. The whole length of a 5 kb rRNA operon was covered by a single read. As the first nanopore-based single molecule sequencer available to researchers, the MinION is an exciting prospect; however, the current error rate limits its ability to compete with existing sequencing technologies, though we do show that MinION sequence reads can enhance contiguity of de novo assembly when used in conjunction with Illumina MiSeq data.

The alternative sigma factor sigma(28) of the extreme thermophile Aquifex aeolicus restores motility to an Escherichia coli fliA mutant.

Sigma factor sigma(28) (sigma(F), FliA, SigD) directs RNA polymerase to transcribe the genes required for flagellar biosynthesis and chemotaxis in many bacteria, including Bacillus subtilis, Legionella pneumophila, Salmonella typhimurium, Escherichia coli, Yersinia enterolytica, Treponema maltophilum and Pseudomonas aeruginosa. Remarkably the fliA gene from the extreme thermophile Aquifex aeolicus restored motility to the E. coli mutant at relatively low temperature, albeit partially. This clearly demonstrates that A. aeolicus sigma(28) is able to direct RNA polymerase to E. coli sigma(28)-dependent promoters and take part in the complex interactions required to support transcription of the flagellar apparatus in vivo. The ability of A. aeolicus sigma(28) to function with mesophilic components shows that critical functional interactions made by these sigma factors are well conserved, and are not dependent upon high temperature. We over-produced and purified the sigma(28) protein and demonstrated binding to E. coli core RNA polymerase in vitro. In common with SigD from B. subtilis, but unlike most sigma factors, A. aeolicus sigma(28) showed DNA binding activity in vitro but there was no evidence of sequence specificity. We note that A. aeolicus sigma(28) is a good candidate for structural studies.

The biology of enhancer-dependent transcriptional regulation in bacteria: insights from genome sequences.

The bacterial transcription factor sigma(N) (sigma-N, sigma-54, RpoN) confers upon RNA polymerase (RNAP) properties distinct from those of the major house-keeping form of RNAP, which contains sigma(70) (sigma-70, RpoD). Transcription by RNAP containing sigma(N) is subject to enhancer-dependent regulation. Far from being an 'oddity' or 'exception to the rule', the occurrence of sigma(N) in the genome sequences of such diverse bacteria as Aquifex aeolicus, Bacillus subtilis, Chlamydia spp. and Borrelia burgdorferi argues for its biological importance. The availability of complete genome sequences of several (eu)bacteria offers an opportunity to extend our understanding of this special form of transcriptional regulation. By scanning their genome sequences, new functions have been predicted for enhancer-dependent transcription in A. aeolicus, Chlamydia trachomatis, Escherichia coli, Treponema pallidum and B. burgdorferi.

Phylogenetic analysis of transformable strains of thermophilic Bacillus species.

Few strains of thermophilic Bacillus spp are readily transformable with plasmid DNA. Given the considerable phylogenetic and phenotypic diversity amongst thermophilic bacilli, we have examined whether transformability is a trait associated with a particular phylogenetic group, by sequencing the 16S ribosomal RNA genes from transformable strains NUB3621, K1041, and NRRL1174. Although all of these strains were described in the literature as B. stearothermophilus, only NRRL1174 is closely related to the type strain of this species. Based on its 16S rDNA sequence and physiological data K1041 appeared to belong to the species B. thermodenitrificans, while NUB3621 showed a slightly closer relationship to B. thermoglucosidasius than to B. stearothermophilus. Therefore we conclude that the trait of transformability, though possibly strain-specific, is not limited to a single species of thermophilic Bacillus.

Salmonella Gallinarum field isolates from laying hens are related to the vaccine strain SG9R.

Salmonella enterica subspecies enterica serotype Gallinarum can cause severe systemic disease in chickens and a live Salmonella Gallinarum 9R vaccine (SG9R) has been used widely to control disease. Using whole-genome sequencing we found point mutations in the pyruvate dehydrogenase (aceE) and/or lipopolysaccharide 1,2-glucosyltransferase (rfaJ) genes that likely explain the attenuation of the SG9R vaccine strain. Molecular typing using Pulsed Field Gel Electrophoresis and Multiple-Locus Variable number of tandem repeat Analysis showed that strains isolated from different layer flocks in multiple countries and the SG9R vaccine strain were similar. The genome of one Salmonella Gallinarum field strain, isolated from a flock with a mortality peak and selected on the basis of identical PFGE and MLVA patterns with SG9R, was sequenced. We found 9 non-silent single-nucleotide differences distinguishing the field strain from the SG9R vaccine strain. Our data show that a Salmonella Gallinarum field strain isolated from laying hens is almost identical to the SG9R vaccine. Mutations in the aceE and rfaJ genes could explain the reversion to a more virulent phenotype. Our results highlight the importance of using well defined gene deletion mutants as vaccines.

Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus.

The genome sequence of the extremely thermophilic bacterium Aquifex aeolicus encodes alternative sigma factor sigma(N) (sigma(54), RpoN) and five potential sigma(N)-dependent transcriptional activators. Although A. aeolicus possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative sigma(N)-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in Escherichia coli), and purified A. aeolicus sigma(N) and the NifA homologue, AQ_218. Purified A. aeolicus sigma(N) bound to E. coli core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative sigma(N)-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus sigma(N) supported A. aeolicus NifA-dependent transcription from the glnHp2 promoter. The E. coli activator PspFDeltaHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnBA operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the sigma(N) enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and sigma(N) have coevolved.

The C-terminal 12 amino acids of sigma(N) are required for structure and function.

The sigma(N) protein is an alternative sigma subunit of bacterial RNA polymerase. We investigated the role of a 12-amino-acid "tail" at the C-terminus of Klebsiella pneumoniae sigma(N), which was predicted to be largely surface-exposed and to be mostly loop (that is not alpha-helical or beta-strand). Deletion of this tail from N-terminal hexahistidine-tagged sigma(N) led to loss of sigma(N)-dependent transcription activity in vivo. We overexpressed and purified this deletion-mutant protein for in vitro characterization. The purified deleted protein showed decreased RNA polymerase core- and DNA-binding activities compared to the full-length protein and transcription activity was greatly impaired. Furthermore, evidence from circular dichroism and protease digestion experiments together suggested that deletion of the C-terminus tail resulted in a loss of conformational constraint in the protein. We discuss a possible structural role for the 12 amino acids at the C-terminus of sigma(N).