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Auto Tag: Comparative genomics

July 7, 2019

Complete genome sequencing and targeted mutagenesis reveal virulence contributions of Tal2 and Tal4b of Xanthomonas translucens pv. undulosa ICMP11055 in bacterial leaf streak of wheat

Bacterial leaf streak caused by Xanthomonas translucens pv. undulosa (Xtu) is an important disease of wheat (Triticum aestivum) and barley (Hordeum vulgare) worldwide. Transcription activator-like effectors (TALEs) play determinative roles in many of the plant diseases caused by the different species and pathovars of Xanthomonas, but their role in this disease has not been characterized. ICMP11055 is a highly virulent Xtu strain from Iran. The aim of this study was to better understand genetic diversity of Xtu and to assess the role of TALEs in bacterial leaf streak of wheat by comparing the genome of this strain to the recently completely sequenced genome of a U.S. Xtu strain, and to several other draft X. translucens genomes, and by carrying out mutational analyses of the TALE (tal) genes the Iranian strain might harbor. The ICMP11055 genome, including its repeat-rich tal genes, was completely sequenced using single molecule, real-time technology (Pacific Biosciences). It consists of a single circular chromosome of 4,561,583 bp, containing 3,953 genes. Whole genome alignment with the genome of the United States Xtu strain XT4699 showed two major re-arrangements, nine genomic regions unique to ICMP11055, and one region unique to XT4699. ICMP110055 harbors 26 non-TALE type III effector genes and seven tal genes, compared to 25 and eight for XT4699. The tal genes occur singly or in pairs across five scattered loci. Four are identical to tal genes in XT4699. In addition to common repeat-variable diresidues (RVDs), the tal genes of ICMP11055, like those of XT4699, encode several RVDs rarely observed in Xanthomonas, including KG, NF, Y*, YD, and YK. Insertion and deletion mutagenesis of ICMP11055 tal genes followed by genetic complementation analysis in wheat cv. Chinese Spring revealed that Tal2 and Tal4b of ICMP11055 each contribute individually to the extent of disease caused by this strain. A largely conserved ortholog of tal2 is present in XT4699, but for tal4b, only a gene with partial, fragmented RVD sequence similarity can be found. Our results lay the foundation for identification of important host genes activated by Xtu TALEs as targets for the development of disease resistant varieties.

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July 7, 2019

Comparative sequence analysis of multidrug-resistant IncA/C plasmids from Salmonella enterica

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.

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July 7, 2019

Comparative genomic and regulatory analyses of natamycin production of Streptomyces lydicus A02.

Streptomyces lydicus A02 is used by industry because it has a higher natamycin-producing capacity than the reference strain S. natalensis ATCC 27448. We sequenced the complete genome of A02 using next-generation sequencing platforms, and to achieve better sequence coverage and genome assembly, we utilized single-molecule real-time (SMRT) sequencing. The assembled genome comprises a 9,307,519-bp linear chromosome with a GC content of 70.67%, and contained 8,888 predicted genes. Comparative genomics and natamycin biosynthetic gene cluster (BGC) analysis showed that BGC are highly conserved among evolutionarily diverse strains, and they also shared closer genome evolution compared with other Streptomyces species. Forty gene clusters were predicted to involve in the secondary metabolism of A02, and it was richly displayed in two-component signal transduction systems (TCS) in the genome, indicating a complex regulatory systems and high diversity of metabolites. Disruption of the phoP gene of the phoR-phoP TCS and nsdA gene confirmed phosphate sensitivity and global negative regulation of natamycin production. The genome sequence and analyses presented in this study provide an important molecular basis for research on natamycin production in Streptomyces, which could facilitate rational genome modification to improve the industrial use of A02.

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July 7, 2019

In silico analysis of protein toxin and bacteriocins from Lactobacillus paracasei SD1 genome and available online databases.

Lactobacillus paracasei SD1 is a potential probiotic strain due to its ability to survive several conditions in human dental cavities. To ascertain its safety for human use, we therefore performed a comprehensive bioinformatics analysis and characterization of the bacterial protein toxins produced by this strain. We report the complete genome of Lactobacillus paracasei SD1 and its comparison to other Lactobacillus genomes. Additionally, we identify and analyze its protein toxins and antimicrobial proteins using reliable online database resources and establish its phylogenetic relationship with other bacterial genomes. Our investigation suggests that this strain is safe for human use and contains several bacteriocins that confer health benefits to the host. An in silico analysis of protein-protein interactions between the target bacteriocins and the microbial proteins gtfB and luxS of Streptococcus mutans was performed and is discussed here.

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July 7, 2019

Extraction of high molecular weight DNA from fungal rust spores for long read sequencing.

Wheat rust fungi are complex organisms with a complete life cycle that involves two different host plants and five different spore types. During the asexual infection cycle on wheat, rusts produce massive amounts of dikaryotic urediniospores. These spores are dikaryotic (two nuclei) with each nucleus containing one haploid genome. This dikaryotic state is likely to contribute to their evolutionary success, making them some of the major wheat pathogens globally. Despite this, most published wheat rust genomes are highly fragmented and contain very little haplotype-specific sequence information. Current long-read sequencing technologies hold great promise to provide more contiguous and haplotype-phased genome assemblies. Long reads are able to span repetitive regions and phase structural differences between the haplomes. This increased genome resolution enables the identification of complex loci and the study of genome evolution beyond simple nucleotide polymorphisms. Long-read technologies require pure high molecular weight DNA as an input for sequencing. Here, we describe a DNA extraction protocol for rust spores that yields pure double-stranded DNA molecules with molecular weight of >50 kilo-base pairs (kbp). The isolated DNA is of sufficient purity for PacBio long-read sequencing, but may require additional purification for other sequencing technologies such as Nanopore and 10× Genomics.

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July 7, 2019

Characterization of a PVL-negative community-acquired methicillin-resistant Staphylococcus aureus strain of sequence type 88 in China.

Sequence type 88 community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strain SR434, isolated from an outpatient with skin and soft tissue infection, was subjected to whole genome sequencing, antimicrobial susceptibility testing, mouse skin infection model and hemolysis analysis to identify its virulence and resistance determinants. MRSA strain SR434 is resistant to clindamycin, erythromycin and fosfomycin. Four plasmids with resistance genes were identified in this strain, including a 20,658bp blaZ-carrying plasmid, a 2473bp ermC-carrying plasmid, a 2622bp fosB7-carrying plasmid (86% identity with plasmid in a ST2590 MRSA strain) and a 4817bp lnuA-carrying plasmid (99% identity with pLNU4 from bovine coagulase-nagetive Staphylococci). This strain contains staphylococcal cassette chromosome mec type IV and does not contain arginine catabolic mobile element or Panton-Valentine-Leukocidin. SR434 harbors genomic islands ?Saa, ?Saß, ?Sa? and FSa3 and pathogenicity islands ?Sa2 that carries genes encoding toxic shock syndrome toxin 1, superantigen enterotoxin C and superantigen enterotoxin L. Mouse skin infection model results show that SR434 had similar virulence potential causing invasive skin infection as a PVL-negative epidemic Korea clone HL1 (ST72). CA-MRSA strain of ST88 lineage might be a great concern for its high virulence. PVL has limited contribution to virulence phenotype among this lineage. Copyright © 2017 Elsevier GmbH. All rights reserved.

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July 7, 2019

Large scale and significant expression from pseudogenes in Sodalis glossinidius – a facultative bacterial endosymbiont

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50% pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple omic strategies, combining: Illumina and Pacific Biosciences Single-Molecule Real Time DNA-sequencing and annotation; stranded RNA-sequencing; and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53% and 74% of the Sodalis transcriptome remains active in cell-free culture. Mean sense transcription from Coding Domain Sequences (CDS) is four-times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40% of the 2,729 genes in the core genome, suggesting are stable and/or Sodalis is a recent introduction across the Glossina genus as a facultative symbiont. These data further shed light on the importance of transcriptional and translational control in deciphering host-microbe interactions, and demonstrate that pseudogenes are more complex than a simple degrading DNA sequence. The combination of genomics, transcriptomics and proteomics give a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.

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July 7, 2019

Draft genome sequence of the halophilic Halobacillus mangrovi KTB 131 isolated from Topan salt of the Jeon-nam in Korea.

The draft genome sequence of the halophilic bacterium Halobacillus mangrovi KTB 131, isolated from Topan salt of the Jeon-nam in Korea, was established. The genome comprises 4,151,649 bp, with a G + C content of 41.6%. The strain displays a high number of genes responsible for secondary metabolite biosynthesis, transport, and catabolism compared to other Halobacillus bacterial genus members. Numerous genes responsible for various transport systems, solute accumulation, and aromatic/sulfur decomposition were detected. The first genomic analysis encourages further research on comparative genomics and potential biotechnological applications. The whole draft genome sequence of Halobacillus mangrovi KTB 131 is now available (Bioproject PRJNA380285).

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July 7, 2019

Comparative genomic analysis of Mycobacterium tuberculosis Beijing-like strains revealed specific genetic variations associated with virulence and drug resistance.

Isolates of the Mycobacterium tuberculosis lineage 2/East-Asian are considered one of the most successful strains due to their increased pathogenicity, hyper-virulence associated with drug resistance, and high transmission. Recent studies in Colombia have shown that the Beijing-like genotype is associated with multidrug-resistance and high prevalence in the southwest of the country, but the genetic basis of its success in dissemination is unknown. In contribution to this matter, we obtained the whole sequences of six genomes of clinical isolates assigned to the Beijing-like genotype. The genomes were compared with the reference genome of M. tuberculosis H37Rv and 53 previously published M. tuberculosis genomes. We found that the six Beijing-like isolates belong to a modern Beijing sub-lineage and share specific genomic variants: i.e. deletion in the PPE8 gene, in Rv3806c (ubiA) responsible of high ethambutol resistance and in Rv3862c (whiB6) which is involved in granuloma formation and virulence, are some of them. Moreover, each isolated has exclusively single nucleotide polymorphisms (SNPs) in genes related with cell wall processes and cell metabolism. We identified polymorphisms in genes related to drug resistance that could explain the drug-resistant phenotypes found in the six isolates from Colombia. We hypothesize that changes due to these genetic variations contribute to the success of these strains. Finally, we analyzed the IS6110 insertion sequences finding very low variance between them, suggesting that SNPs is the major cause of variability found in Beijing-like strains circulating in Colombia. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Genomic analysis of Clavibacter michiganensis reveals insight into virulence strategies and genetic diversity of a gram-positive bacterial pathogen.

Clavibacter michiganensis subsp. michiganensis is a gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial canker disease. In this study, we sequenced and assembled genomes of 11 C. michiganensis subsp. michiganensis strains isolated from infected tomato fields in California as well as five Clavibacter strains that colonize tomato endophytically but are not pathogenic in this host. The analysis of the C. michiganensis subsp. michiganensis genomes supported the monophyletic nature of this pathogen but revealed genetic diversity among strains, consistent with multiple introduction events. Two tomato endophytes that clustered phylogenetically with C. michiganensis strains capable of infecting wheat and pepper and were also able to cause disease in these plants. Plasmid profiles of the California strains were variable and supported the essential role of the pCM1-like plasmid and the CelA cellulase in virulence, whereas the absence of the pCM2-like plasmid in some pathogenic C. michiganensis subsp. michiganensis strains revealed it is not essential. A large number of secreted C. michiganensis subsp. michiganensis proteins were carbohydrate-active enzymes (CAZymes). Glycome profiling revealed that C. michiganensis subsp. michiganensis but not endophytic Clavibacter strains is able to extensively alter tomato cell-wall composition. Two secreted CAZymes found in all C. michiganensis subsp. michiganensis strains, CelA and PelA1, enhanced pathogenicity on tomato. Collectively, these results provide a deeper understanding of C. michiganensis subsp. michiganensis diversity and virulence strategies.

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July 7, 2019

Comparative genome analysis of the Flavobacteriales bacterium strain UJ101, isolated from the gut of Atergatis reticulatus.

Here we report the comparative genomic analysis of strain UJ101 with 15 strains from the family Flavobacteriaceae, using the CGExplorer program. Flavobacteriales bacterium strain UJ101 was isolated from a xanthid crab, Atergatis reticulatus, from the East Sea near Korea. The complete genome of strain UJ101 is a 3,074,209 bp, single, circular chromosome with 30.74% GC content. While the UJ101 genome contains a number of annotated genes for many metabolic pathways, such as the Embden-Meyerhof pathway, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle, genes for the Entner-Douddoroff pathway are not found in the UJ101 genome. Overall, carbon fixation processes were absent but nitrate reduction and denitrification pathways were conserved. The UJ101 genome was compared to genomes from other marine animals (three invertebrate strains and 5 fish strains) and other marine animal- derived genera. Notable results by genome comparisons showed that UJ101 is capable of denitrification and nitrate reduction, and that biotin-thiamine pathway participation varies among marine bacteria; fish-dwelling bacteria, freeliving bacteria, invertebrate-dwelling bacteria, and strain UJ101. Pan-genome analysis of the 16 strains in this study included 7,220 non-redundant genes that covered 62% of the pan-genome. A core-genome of 994 genes was present and consisted of 8% of the genes from the pan-genome. Strain UJ101 is a symbiotic hetero-organotroph isolated from xanthid crab, and is a metabolic generalist with nitrate-reducing abilities but without the ability to synthesize biotin. There is a general tendency of UJ101 and some fish pathogens to prefer thiamine-dependent glycolysis to gluconeogenesis. Biotin and thiamine auxotrophy or prototrophy may be used as important markers in microbial community studies.

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July 7, 2019

Characterization of the emerging zoonotic pathogen Arcobacter thereius by whole genome sequencing and comparative genomics.

Four Arcobacter species have been associated with human disease, and based on current knowledge, these Gram negative bacteria are considered as potential food and waterborne zoonotic pathogens. At present, only the genome of the species Arcobacter butzleri has been analysed, and still little is known about their physiology and genetics. The species Arcobacter thereius has first been isolated from tissue of aborted piglets, duck and pig faeces, and recently from stool of human patients with enteritis. In the present study, the complete genome and analysis of the A. thereius type strain LMG24486T, as well as the comparative genome analysis with 8 other A. thereius strains are presented. Genome analysis revealed metabolic pathways for the utilization of amino acids, which represent the main source of energy, together with the presence of genes encoding for respiration-associated and chemotaxis proteins. Comparative genome analysis with the A. butzleri type strain RM4018 revealed a large correlation, though also unique features. Furthermore, in silico DDH and ANI based analysis of the nine A. thereius strains disclosed clustering into two closely related genotypes. No discriminatory differences in genome content nor phenotypic behaviour were detected, though recently the species Arcobacter porcinus was proposed to encompass part of the formerly identified Arcobacter thereius strains. The report of the presence of virulence associated genes in A. thereius, the presence of antibiotic resistance genes, verified by in vitro susceptibility testing, as well as other pathogenic related relevant features, support the classification of A. thereius as an emerging pathogen.

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July 7, 2019

Recombination of virulence genes in divergent Acidovorax avenae strains that infect a common host.

Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) pathogenicity island. Ever since the recombination, the cluster has been conserved by strong purifying selection, hinting at its selective importance. Together our analyses suggest that BED is an ancient disease that may owe its host specificity to a highly conserved cluster of 11 T3SS genes.

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July 7, 2019

The genome of the cotton bacterial blight pathogen Xanthomonas citri pv. malvacearum strain MSCT1.

Xanthomonas citri pv. malvacearum is a major pathogen of cotton, Gossypium hirsutum L.. In this study we report the complete genome of the X. citri pv. malvacearum strain MSCT1 assembled from long read DNA sequencing technology. The MSCT1 genome is the first X. citri pv. malvacearum genome with complete coding regions for X. citri pv. malvacearum transcriptional activator-like effectors. In addition functional and structural annotations are presented in this study that will provide a foundation for future pathogenesis studies with MSCT1.

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July 7, 2019

Complete genome sequence of Actinomadura parvosata Subsp. kistnae, a rich source of novel natural product (bio-)chemistry.

The soil dwelling actinomycete strain Actinomadura parvosata subsp. kistnae is the producer of the antiviral antibiotics kistamicin A and B. Genome sequencing and bioinformatic analysis revealed the presence of the kistamycin biosynthetic gene cluster responsible for the formation of these non-ribosomal peptides as well as an impressive number of yet uncharacterized biosynthetic pathways. This includes polyketide, ribosomal and non-ribosomal peptide and a large number of terpenoid biosynthetic loci encoding yet unknown natural products. The genomic data of this strain is thus a treasure trove for genome mining for novel functional metabolites and new biocatalysts.

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