The hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) strain WCUH29 has been intensively and widely used as a model system for identification and evaluation of novel antibacterial targets and pathogenicity. In this announcement, we report the complete genome sequence of HA-MRSA WCUH29 (NCIMB 40771).Copyright © 2019 Lei et al.
Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.Copyright © 2019 American Society for Microbiology.
The bacteriophage exclusion (BREX) system is a novel prokaryotic defense system against bacteriophages. To our knowledge, no study has systematically characterized the function of the BREX system in lactic acid bacteria. Lactobacillus casei Zhang is a probiotic bacterium originating from koumiss. By using single-molecule real-time sequencing, we previously identified N6-methyladenine (m6A) signatures in the genome of L. casei Zhang and a putative methyltransferase (MTase), namely, pglX This work further analyzed the genomic locus near the pglX gene and identified it as a component of the BREX system. To decipher the biological role of pglX, an L. casei Zhang pglX mutant (?pglX) was constructed. Interestingly, m6A methylation of the 5′-ACRCAG-3′ motif was eliminated in the ?pglX mutant. The wild-type and mutant strains exhibited no significant difference in morphology or growth performance in de Man-Rogosa-Sharpe (MRS) medium. A significantly higher plasmid acquisition capacity was observed for the ?pglX mutant than for the wild type if the transformed plasmids contained pglX recognition sites (i.e., 5′-ACRCAG-3′). In contrast, no significant difference was observed in plasmid transformation efficiency between the two strains when plasmids lacking pglX recognition sites were tested. Moreover, the ?pglX mutant had a lower capacity to retain the plasmids than the wild type, suggesting a decrease in genetic stability. Since the Rebase database predicted that the L. casei PglX protein was bifunctional, as both an MTase and a restriction endonuclease, the PglX protein was heterologously expressed and purified but failed to show restriction endonuclease activity. Taken together, the results show that the L. casei Zhang pglX gene is a functional adenine MTase that belongs to the BREX system.IMPORTANCELactobacillus casei Zhang is a probiotic that confers beneficial effects on the host, and it is thus increasingly used in the dairy industry. The possession of an effective bacterial immune system that can defend against invasion of phages and exogenous DNA is a desirable feature for industrial bacterial strains. The bacteriophage exclusion (BREX) system is a recently described phage resistance system in prokaryotes. This work confirmed the function of the BREX system in L. casei and that the methyltransferase (pglX) is an indispensable part of the system. Overall, our study characterizes a BREX system component gene in lactic acid bacteria. Copyright © 2019 American Society for Microbiology.
The first draft genome sequence of the pearl millet blast pathogen Magnaporthe grisea PMg_Dl from India is presented. The genome information of M. grisea will be useful to understand the Magnaporthe speciation, genetic diversity, environmental adaptation, and pathogenic and host range determinants.Copyright © 2019 Prakash et al.
The type VI secretion system (T6SS) facilitates lethal competition between bacteria through direct contact. Comparative genomics has facilitated the study of these systems in Vibrio fischeri, which colonizes the squid host Euprymna scolopes Here, we report the draft genome sequences of two lethal V. fischeri strains that encode the T6SS, FQ-A001 and ES401.Copyright © 2019 Bultman et al.
Halorubrum sp. strain BOL3-1 was isolated from Salar de Uyuni, Bolivia, and sequenced using single-molecule real-time sequencing. Its 3.7-Mbp genome was analyzed for gene content and methylation patterns and incorporated into the Haloarchaeal Genomes Database (http://halo.umbc.edu). The polyextremophilic character and high-elevation environment make the microbe of interest for astrobiology. Copyright © 2019 DasSarma et al.
Scutellaria baicalensis is a well-known medicinal plant that produces biologically active flavonoids, such as baicalin, baicalein, and wogonin. Pharmacological studies have shown that these compounds have anti-inflammatory, anti-bacterial, and anti-cancer activities. Therefore, it is of great significance to investigate the genetic information of S. baicalensis, particularly the genes related to the biosynthetic pathways of these compounds. Here, we constructed the full-length transcriptome of S. baicalensis using a hybrid sequencing strategy and acquired 338,136 full-length sequences, accounting for 93.3% of the total reads. After the removal of redundancy and correction with Illumina short reads, 75,785 nonredundant transcripts were generated, among which approximately 98% were annotated with significant hits in the protein databases, and 11,135 sequences were classified as lncRNAs. Differentially expressed gene (DEG) analysis showed that most of the genes related to flavonoid biosynthesis were highly expressed in the roots, consistent with previous reports that the flavonoids were mainly synthesized and accumulated in the roots of S. baicalensis. By constructing unique transcription models, a total of 44,071 alternative splicing (AS) events were identified, with intron retention (IR) accounting for the highest proportion (44.5%). A total of 94 AS events were present in five key genes related to flavonoid biosynthesis, suggesting that AS may play important roles in the regulation of flavonoid biosynthesis in S. baicalensis. This study provided a large number of highly accurate full-length transcripts, which represents a valuable genetic resource for further research of the molecular biology of S. baicalensis, such as the development, breeding, and biosynthesis of active ingredients.
USA300 is a predominant community-associated methicillin-resistant Staphylococcus aureus strain causing significant morbidity and mortality in North America. We present the full annotated genome sequences of two methicillin-resistant Staphylococcus aureus isolates related to the USA300 pulsotype with the goal of studying the evolutionary relationships of this highly successful strain type.Copyright © 2019 McClure and Zhang.
Avocado (Persea americana Mill.) is an economically important crop because of its high nutritional value. However, the absence of a sequenced avocado reference genome has hindered investigations of secondary metabolism. For next-generation high-throughput transcriptome sequencing, we obtained 365,615,152 and 348,623,402 clean reads as well as 109.13 and 104.10 Gb of sequencing data for avocado mesocarp and seed, respectively, during five developmental stages. High-quality reads were assembled into 100,837 unigenes with an average length of 847.40 bp (N50 = 1725 bp). Additionally, 16,903 differentially expressed genes (DEGs) were detected, 17 of which were related to carotenoid biosynthesis. The expression levels of most of these 17 DEGs were higher in the mesocarp than in the seed during five developmental stages. In this study, the avocado mesocarp and seed transcriptome were also sequenced using single-molecule long-read sequencing to acquired 25.79 and 17.67 Gb clean data, respectively. We identified 233,014 and 238,219 consensus isoforms in avocado mesocarp and seed, respectively. Furthermore, 104 and 59 isoforms were found to correspond to the putative 11 carotenoid biosynthetic-related genes in the avocado mesocarp and seed, respectively. The isoform numbers of 10 out of the putative 11 genes involved in the carotenoid biosynthetic pathway were higher in the mesocarp than those in the seed. Besides, alpha- and beta-carotene contents in the avocado mesocarp and seed during five developmental stages were also measured, and they were higher in the mesocarp than in the seed, which validated the results of transcriptome profiling. Gene expression changes and the associated variations in gene dosage could influence carotenoid biosynthesis. These results will help to further elucidate carotenoid biosynthesis in avocado.
We present the draft genome sequence of an isogenic haploid strain, IR-2idA30(MATa), established from Saccharomyces cerevisiae IR-2. Assembly of long reads and previously obtained contigs from the genome of diploid IR-2 resulted in 50 contigs, and the variations and sequencing errors were corrected by short reads. Copyright © 2019 Fujimori et al.
Eucommia ulmoides Oliver is widely distributed in China. This species has been used mainly in medicine due to the high concentration of chlorogenic acid (CGA), flavonoids, lignans, and other compounds in the leaves and barks. However, the categories of metabolites, dynamic changes in metabolite accumulation and overall molecular mechanisms involved in metabolite biosynthesis during E. ulmoides leaf growth and development remain unknown. Here, a total of 515 analytes, including 127 flavonoids, 46 organic acids, 44 amino acid derivatives, 9 phenolamides, and 16 vitamins, were identified from four E. ulmoides samples using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) (for widely targeted metabolites). The accumulation of most flavonoids peaked in growing leaves, followed by old leaves. UPLC-MS analysis indicated that CGA accumulation increased steadily to a high concentration during leaf growth and development, and rutin showed a high accumulation level in leaf buds and growing leaves. Based on single-molecule long-read sequencing technology, 69,020 transcripts and 2880 novel loci were identified in E. ulmoides. Expression analysis indicated that isoforms in the flavonoid biosynthetic pathway and flavonoid metabolic pathway were highly expressed in growing leaves and old leaves. Co-expression network analysis suggested a potential direct link between the flavonoid and phenylpropanoid biosynthetic pathways via the regulation of transcription factors, including MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic/helix-loop-helix). Our study predicts dynamic metabolic models during leaf growth and development and will support further molecular biological studies of metabolite biosynthesis in E. ulmoides. In addition, our results significantly improve the annotation of the E. ulmoides genome.
Alternative splicing of pre-mRNAs is a crucial mechanism for maintaining protein diversity in eukaryotes without requiring a considerable increase of genes in the number. Due to rapid advances in high-throughput sequencing technologies and computational algorithms, it is anticipated that alternative splicing events will be more intensively studied to address different kinds of biological questions. The occurrences of alternative splicing mean that all exons could be classified to be either constitutively or alternatively spliced depending on whether they are virtually included into all mature mRNAs. From an evolutionary point of view, therefore, the alternatively spliced exons would have been associated with distinctive biological characteristics in comparison with constitutively spliced exons. In this paper, we first outline the representative types of alternative splicing events and exon classification, and then review sequence and evolutionary features for the alternatively spliced exons. The main purpose is to facilitate understanding of the biological implications of alternative splicing in eukaryotes. This knowledge is also helpful to establish computational approaches for predicting the splicing pattern of exons.
Transposable elements (TEs) are agents of genetic variability in phytopathogens as they are a source of adaptive evolution through genome diversification. Although many studies have uncovered information on TEs, the exact mechanism behind TE-induced changes within the genome remains poorly understood. Furthermore, convergent trends towards bigger genomes, emergence of novel genes and gain or loss of genes implicate a TE-regulated genome plasticity of fungal phytopathogens. TEs are able to alter gene expression by revamping the cis-regulatory elements or recruiting epigenetic control. Recent findings show that TEs recruit epigenetic control on the expression of effector genes as part of the coordinated infection strategy. In addition to genome plasticity and diversity, fungal pathogenicity is an area of economic concern. A survey of TE distribution suggests that their proximity to pathogenicity genes TEs may act as sites for emergence of novel pathogenicity factors via nucleotide changes and expansion or reduction of the gene family. Through a systematic survey of literature, we were able to conclude that the role of TEs in fungi is wide: ranging from genome plasticity, pathogenicity to adaptive behavior in evolution. This review also identifies the gaps in knowledge that requires further elucidation for a better understanding of TEs’ contribution to genome architecture and versatility.
Klebsiella pneumoniae 2N3 is a strain of gram-negative bacteria that can degrade chlorimuron-ethyl and grow with chlorimuron-ethyl as the sole nitrogen source. The complete genome of Klebsiella pneumoniae 2N3 was sequenced using third generation high-throughput DNA sequencing technology. The genomic size of strain 2N3 was 5.32 Mb with a GC content of 57.33% and a total of 5156 coding genes and 112 non-coding RNAs predicted. Two hydrolases expressed by open reading frames (ORFs) 0934 and 0492 were predicted and experimentally confirmed by gene knockout to be involved in the degradation of chlorimuron-ethyl. Strains of ?ORF 0934, ?ORF 0492, and wild type (WT) reached their highest growth rates after 8-10 hours in incubation. The degradation rates of chlorimuron-ethyl by both ?ORF 0934 and ?ORF 0492 decreased in comparison to the WT during the first 8 hours in culture by 25.60% and 24.74%, respectively, while strains ?ORF 0934, ?ORF 0492, and the WT reached the highest degradation rates of chlorimuron-ethyl in 36 hours of 74.56%, 90.53%, and 95.06%, respectively. This study provides scientific evidence to support the application of Klebsiella pneumoniae 2N3 in bioremediation to control environmental pollution.
Deinococcus wulumuqiensis 479 (formerly known as Deinococcus radiodurans 479) is the original source strain for the restriction enzyme DrdI. Its complete sequence and full methylome were determined using Pacific Biosciences single-molecule real-time (SMRT) sequencing. Copyright © 2019 Fomenkov et al.
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