Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa.

BACKGROUND: Bacillus endophyticus is a soil plant-endophytic bacterium, while B. anthracis is the causative agent of anthrax. The virulence factors of B. anthracis are the plasmid encoded tripartite toxins (pXO1) and poly-γ-glutamic acid (PGA) capsule (pXO2). B. endophyticus isolated alongside B. anthracis from animals that died of anthrax in Northern Cape Province (NCP), South Africa, harbored polyglutamate genes. The study compared the characteristics of B. anthracis and B. endophyticus with other Bacillus species with a focus on the presence of the PGA capsule or/and unbound PGA. The morphology and whole genome sequence analysis of B. endophyticus strains and B. anthracis were compared. RESULTS: In conventional microbiology, B. endophyticus showed gram-positive round-shaped rods in single/short chains, which were endospore-forming, non-motile, non-haemolytic with white and dry colonies, and γ-phage resistant. B. anthracis was differentiated from B. endophyticus based on the latter's box-shaped rods in pairs/long chains, white-grey and slimy colonies, encapsulated and γ-phage susceptible. The study identified a PGA polyglutamate synthase operon that consisted of pgsBCA, γ-glutamyltranspeptidase (ggt) and pgsE in B. endophyticus genomes. CONCLUSIONS: PGA regions of B. anthracis contain capBCADE genes located in the pXO2 required for capsulation formation, while B. endophyticus contain the pgsBCAE genes in the chromosome. Whole genome and microbiology analysis identified B. endophyticus, as a non-capsuled endospore-forming bacterium that consists of PGA required for biosynthesis. B. endophyticus strains do not synthesize surface associated PGA, therefore capsule visualization of B. anthracis is a key diagnostic characteristic. The study highlights the significance of using whole genome shotgun sequencing to identify virulence and other important genes that might be present amongst unknown samples from natural outbreaks. None of the B. anthracis related plasmids or virulence genes were found in the B. endophyticus genomes.

Humidity modifies species-specific and age-dependent heat stress effects in an insect host-parasitoid interaction.

Climate change is projected to increase the frequency and intensity of extreme heat events, and may increase humidity levels, leading to coupled thermal and hydric stress. However, how humidity modulates the impacts of heat stress on species and their interactions is currently unknown. Using an insect host-parasitoid interaction: the Indian meal moth, Plodia interpunctella, and its endoparasitoid wasp, Venturia canescens, we investigated how humidity interacted with heat stress duration, applied at different host developmental stages, to affect life history traits. Hosts parasitized as 4th instar larvae and unparasitized hosts were maintained in high- (60.8% RH) or low-humidity (32.5% RH) at constant 28°C. They were then exposed to a 38°C thermal stress with a duration of 0 (no heat stress), 6 or 72 h in either the 4th or 5th host instar. Neither humidity nor heat stress duration affected emergence of unparasitized hosts, but increasing heat stress duration during the 4th instar decreased parasitoid emergence irrespective of humidity. When applied during the 5th instar, increasing heat duration decreased parasitoid emergence under low humidity, but no effect of heat stress was found under high humidity. Moreover, experiencing longer heat stress in the 4th instar increased host larval development time and decreased body size under high humidity, but this effect differed under low humidity; increasing heat duration in the 5th instar decreased parasitoid body sizes only under low humidity. Larval stage and heat stress duration directly affected parasitized host survival time, with a concomitant indirect reduction of parasitoid sizes. We show that humidity modifies key life history responses of hosts and parasitoids to heat stress in species-specific ways, highlighting the potential importance of humidity in regulating host-parasitoid interactions and their population dynamics. Finally, we emphasize that interactions between environmental stressors need to be considered in climate change research.

De-regulation of the RBBP6 isoform 3/DWNN in human cancers.

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. The human RBBP6 gene codes for three protein isoforms and isoform 3 consists of the domain with no name domain only whilst the other two isoforms, 1 and 2 comprise of additional zinc, RING, retinoblastoma and p53 binding domains. In this study, the localization of RBBP6 using RBBP6 variant 3 mRNA-specific probe was performed to investigate the expression levels of the gene in different tumours and find a link between RBBP6 and human carcinogenesis. Using FISH, real-time PCR and Western blotting analysis our results show that RBBP6 isoform 3 is down-regulated in human cancers. RBBP6 isoform 3 knock-down resulted in reduced G2/M cell cycle arrest whilst its over-expression resulted in increased G2/M cell cycle arrest using propidium iodide DNA staining. The results further demonstrate that the RBBP6 isoform 3 may be the cell cycle regulator and involved in mitotic apoptosis not the isoform 1 as previously reported for mice. In conclusion, these findings suggest that RBBP6 isoform 3 is a cell cycle regulator and may be de-regulated in carcinogenesis.

Full genomic sequence of an African avian paramyxovirus type 4 strain isolated from a wild duck.

A random amplification/deep sequencing approach was applied to determine the complete genomic sequence of an Avian Paramyxovirus Type 4 (APMV-4) strain isolated from a wild duck in South Africa in 2010. This sequence represents the fourth full genome of APMV-4 in public sequence databases and the first for the African continent. A total of 87,402,081 Illumina paired-end reads were obtained of which 47,338,867 (54.16 %) mapped to the reference genome EU877976. The entire genomic sequence of 15,054 nt, including the intact termini, was recovered at a high redundancy (coverage per base: average = 198,861.06, minimum = 52 and maximum = 1,790,889). Pairwise comparison of full genomic nucleotide sequences indicated that APMV-4/Egyptian goose/South Africa/N1468/10 shared 97.3 % sequence identity with APMV-4/KR/YJ/06, 96.4 % sequence identity with APMV-4/mallard/Belgium/15129/07 and 90.8 % nucleotide sequence identity with APMV-4/duck/HK/D3/75. Genomic features were consistent with previously sequenced viruses including predicted open reading frames for the NP, P, F and L genes, but variations in coding regions for the M and HN genes were identified. The sequencing approach adopted in this study could successfully indicate quasispecies in the viral stock.

A low-cost flow cytometric assay for the detection and quantification of apoptosis using an anionic halogenated fluorescein dye.

We describe here a technical improvement of an established colorimetric method used to detect and measure the occurrence of apoptosis in mammalian cells during in vitro cell culture. This assay uses an anionic halogenated fluorescein dye that is taken up by apoptotic cells at the stage of phosphatidylserine externalization. We demonstrate that apoptotic cells stained with this dye can be detected by flow cytometric analysis. Furthermore, we show that the modified method compares well with the standard annexin-V-based apoptosis assay and that it is significantly more cost-effective than the annexin-V assay.

The early transcriptome response of cassava (Manihot esculenta Crantz) to mealybug (Phenacoccus manihoti) feeding.

The mealybug, Phenacoccus manihoti, is a leading pest of cassava (Manihot esculenta Crantz), damaging this crop globally. Although the biological control of this mealybug using natural predators has been established, resistance breeding remains an important means of control. Understanding plant responses to insect herbivory, by determining and identifying differentially expressed genes (DEGs), is a vital step towards the understanding of molecular mechanisms of defence responses in plants and the development of resistant cultivars by gene editing. Morphological and molecular analysis confirmed the mealybug identity as Phenacoccus manihoti (Matile-Ferrero). The transcriptome response of the green mite resistant cassava genotype AR23.1 was compared to P40/1 with no known resistance at 24 and 72 hours of mealybug infestation compared to non-infested mock. A total of 301 and 206 genes were differentially expressed at 24 and 72 of mealybug infestation for AR23.1 and P40/1 genotypes respectively, using a log2 fold change and P-value ≤ 0.05. Gene ontology functional classification revealed an enrichment of genes in the secondary metabolic process category in AR23.1 in comparison with P40/1, while genes in the regulation of molecular function, cellular component biogenesis and electron carrier categories were more significantly enriched in P40/1 than in AR23.1. Biological pathway analysis, based on KEGG, revealed a significant enrichment of plant-pathogen interaction and plant hormonal signal transduction pathways for a cohort of up-regulated and down-regulated DEGs in both genotypes. Defence-related genes such as 2-oxogluterate, gibberellin oxidase and terpene synthase proteins were only induced in genotype AR23.1 and not in P40/1, and subsequently validated by RT-qPCR. The study revealed a difference in response to mealybug infestation in the two genotypes studied, with AR23.1 showing a higher number of differentially expressed transcripts post mealybug infestation at 24 and 72 hours. Candidate defence-related genes that were overexpressed in the AR23.1 genotype post mealybug infestation will be useful in future functional studies towards the control of mealybugs.

A draft genome sequence of the rose black spot fungus Diplocarpon rosae reveals a high degree of genome duplication.

BACKGROUND: Black spot is one of the most severe and damaging diseases of garden roses. We present the draft genome sequence of its causative agent Diplocarpon rosae as a working tool to generate molecular markers and to analyze functional and structural characteristics of this fungus. RESULTS: The isolate DortE4 was sequenced with 191x coverage of different read types which were assembled into 2457 scaffolds. By evidence supported genome annotation with the MAKER pipeline 14,004 gene models were predicted and transcriptomic data indicated that 88.5% of them are expressed during the early stages of infection. Analyses of k-mer distributions resulted in unexpectedly large genome size estimations between 72.5 and 91.4 Mb, which cannot be attributed to its repeat structure and content of transposable elements alone, factors explaining such differences in other fungal genomes. In contrast, different lines of evidences demonstrate that a huge proportion (approximately 80%) of genes are duplicated, which might indicate a whole genome duplication event. By PCR-RFLP analysis of six paralogous gene pairs of BUSCO orthologs, which are expected to be single copy genes, we could show experimentally that the duplication is not due to technical error and that not all isolates tested possess all of the paralogs. CONCLUSIONS: The presented genome sequence is still a fragmented draft but contains almost the complete gene space. Therefore, it provides a useful working tool to study the interaction of D. rosae with the host and the influence of a genome duplication outside of the model yeast in the background of a phytopathogen.

Significant up-regulation of 1-ACBP, B-ACBP and PBR genes in immune cells within the oesophageal malignant tissue and a possible link in carcinogenic angiogenesis.

Oesophageal cancer ranks as the sixth most common malignancy in the world, and recent evidence has shown that its incidence is increasing. ACBPs (Acyl-coA binding proteins) act as intracellular carrier-proteins for medium to long chain acyl-coA, mediating fatty acid transport to the mitochondrion for ß-oxidation. ACBPs are also believed to be putative ligands of PBR (peripheral benzodiazepine receptor), and once they bind to this receptor they facilitate mitochondrial membrane permeabilization, presumably favouring apoptosis. The main aim of the study was to establish the expression patterns of 1- Acyl-coA binding proteins (1-ACBP), B- Acyl-coA binding proteins (B-ACBP), and peripheral bezodiazepine receptor (PBR) in oesophageal cancer, and to link their roles with the disease. In situ hybridization and quantitative real-time PCR methods were performed to determine localization and the expression levels of the three genes in oesophageal cancer. All three genes illustrated substantial up-regulation within the malignant tissue sections as compared to normal oesophageal sections, all three transcripts localized specifically to mast cells, plasma cells and lymphocytes in diseased and normal tissue section. In the diseased tissue B-ACBP and 1-ACBP mRNA localized to endothelial cells of blood vessels in the submucosa. B-ACBP also localized to the nucleus of squamous epithelial cells. PBR localization was indicated in tumour islands of invasive tissue sections. Quantitative RT-PCR also indicated that the expression levels of PBR were higher as compared to the ACBP genes expression in tumours. These results show that 1-ACBP, B-ACBP and PBR play a role in the pathogenesis of oesophageal tumours and possibly in carcinogenic angiogenesis.

Whole-genome sequencing for an enhanced understanding of genetic variation among South Africans.

The Southern African Human Genome Programme is a national initiative that aspires to unlock the unique genetic character of southern African populations for a better understanding of human genetic diversity. In this pilot study the Southern African Human Genome Programme characterizes the genomes of 24 individuals (8 Coloured and 16 black southeastern Bantu-speakers) using deep whole-genome sequencing. A total of ~16 million unique variants are identified. Despite the shallow time depth since divergence between the two main southeastern Bantu-speaking groups (Nguni and Sotho-Tswana), principal component analysis and structure analysis reveal significant (p < 10-6) differentiation, and FST analysis identifies regions with high divergence. The Coloured individuals show evidence of varying proportions of admixture with Khoesan, Bantu-speakers, Europeans, and populations from the Indian sub-continent. Whole-genome sequencing data reveal extensive genomic diversity, increasing our understanding of the complex and region-specific history of African populations and highlighting its potential impact on biomedical research and genetic susceptibility to disease.

Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho.

INTRODUCTION: Bacillus anthracis is the causative agent of anthrax, a disease endemic in regions of Northern Cape Province and Kruger National Park of South Africa. Accurate identification of virulent B. anthracis is essential but challenging due to its close relationship with other members of B. cereus group. This study characterized B. anthracis and Bacillus species that were recovered from animals and the environment where animals died of anthrax symptoms in southern Africa using a polyphasic approach. METHODOLOGY: For this purpose, 3 B. anthracis and 10 Bacillus isolates were subjected to microbiology tests, BiologOmniLog identification system (Biolog), 16S ribosomal RNA (rRNA) sequence analysis, polymerase chain reaction (PCR) detection of protective antigen (pag) and capsule (cap) regions, and real-time PCR using hybridization probes targeting chromosomal, pag, and capC genes. RESULTS: The Bacillus isolates were non-hemolytic, non-motile, and susceptible to penicillin, which is typical of B. anthracis, but resistant to gamma phage, unlike typical B. anthracis. The Biolog system and 16S rRNA gene sequence analysis identified most of the Bacillus isolates as B. endophyticus (7 of 10). Conventional PCR revealed that most of the Bacillus isolates contained capBCA gene regions. This highlights the limitation of the specificity of conventional PCR and the fact that the real-time PCR is more specific and reliable for anthrax diagnosis. CONCLUSIONS: Real-time PCR, 16S rRNA sequencing, and confirmatory microbiology tests including phage resistance distinguished Bacillus isolates from B. anthracis in this study. Identification of B. anthracis should be done using a polyphasic approach.

Draft Genome Sequences of Two South African Bacillus anthracis Strains.

Bacillus anthracis is a Gram-positive bacterium that causes anthrax, mainly in herbivores through exotoxins and capsule produced on plasmids, pXO1 and pXO2. This paper compares the whole-genome sequences of two B. anthracis strains from an endemic region and a sporadic outbreak in South Africa. Sequencing was done using next-generation sequencing technologies.

Complete Genome Sequence of Mannheimia haemolytica Strain Mh10517, Isolated from Sheep in South Africa.

Respiratory disease caused by Mannheimia haemolytica is a major concern in the cattle and small stock industry worldwide. This problem arises due to the interaction of numerous contributing factors, including physical stresses associated with weaning, shipment, inclement weather, and overcrowding coupled with viral and bacterial infections. The whole genome of M. haemolytica strain Mh10517 was analyzed using an Illumina MiSeq high-throughput sequencing platform. The genome size is 2.67 Mb with 2,879 predicted gene sequences. The availability of this genome sequence will advance studies on various aspects of the biology of M. haemolytica in Africa and the world at large.

Draft Genome Sequence of the Antarctic Polyextremophile Nesterenkonia sp. Strain AN1.

Nesterenkonia sp. strain AN1 was isolated from Antarctic soil and is a polyextremophile, being tolerant of low temperatures, high salt concentrations, and high alkalinity. Here we report the draft genome sequence of this strain.

Genome-wide SNP detection, validation, and development of an 8K SNP array for apple.

As high-throughput genetic marker screening systems are essential for a range of genetics studies and plant breeding applications, the International RosBREED SNP Consortium (IRSC) has utilized the Illumina Infinium® II system to develop a medium- to high-throughput SNP screening tool for genome-wide evaluation of allelic variation in apple (Malus×domestica) breeding germplasm. For genome-wide SNP discovery, 27 apple cultivars were chosen to represent worldwide breeding germplasm and re-sequenced at low coverage with the Illumina Genome Analyzer II. Following alignment of these sequences to the whole genome sequence of 'Golden Delicious', SNPs were identified using SoapSNP. A total of 2,113,120 SNPs were detected, corresponding to one SNP to every 288 bp of the genome. The Illumina GoldenGate® assay was then used to validate a subset of 144 SNPs with a range of characteristics, using a set of 160 apple accessions. This validation assay enabled fine-tuning of the final subset of SNPs for the Illumina Infinium® II system. The set of stringent filtering criteria developed allowed choice of a set of SNPs that not only exhibited an even distribution across the apple genome and a range of minor allele frequencies to ensure utility across germplasm, but also were located in putative exonic regions to maximize genotyping success rate. A total of 7867 apple SNPs was established for the IRSC apple 8K SNP array v1, of which 5554 were polymorphic after evaluation in segregating families and a germplasm collection. This publicly available genomics resource will provide an unprecedented resolution of SNP haplotypes, which will enable marker-locus-trait association discovery, description of the genetic architecture of quantitative traits, investigation of genetic variation (neutral and functional), and genomic selection in apple.