June 1, 2021  |  

Genome analysis of a bacterium that causes lameness.

Lameness is a significant problem resulting in millions of dollars in lost revenue annually. In commercial broilers, the most common cause of lameness is bacterial chondronecrosis with osteomyelitis (BCO). We are using a wire flooring model to induce lameness attributable to BCO. We used 16S ribosomal DNA sequencing to determine that Staphylococcus spp. were the main species associated with BCO. Staphylococcus agnetis, which previously had not been isolated from poultry, was the principal species isolated from the majority of the bone lesion samples. Administering S. agnetis in the drinking water to broilers reared on wire flooring increased the incidence of BCO three-fold when compared with broilers drinking tap water (P = 0.001). We found that the minimum effective dose of Staphylococcus agnetis to induce BCO in broilers grown on wire flooring experiment is 105 cfu/ml. We used PacBio and Illumina sequencing to assemble a 2.4 Mbp contig representing the genome and a 34 kbp contig for the largest plasmid of S. agnetis. Annotation of this genome is underway through comparative genomics with other Staphylococcus genomes, and identification of virulence factors. Our goal is to elucidate genetic diversity, toxins, and pathogenicity determinants, for this poorly characterized species. Isolating pathogenic bacterial species, defining their likely route of transmission to broilers, and genomic analyses will contribute substantially to the development of measures for mitigating BCO losses in poultry.


June 1, 2021  |  

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence, DNA methylation is characterized in the process of sequencing. PacBio® sequencing has also been applied to microbial transcriptomes. Long reads enable sequencing of full-length cDNAs allowing for identification of complete gene and operon sequences without the need for transcript assembly. We will highlight several examples where these capabilities have been leveraged in the areas of industrial microbiology, including biocommodities, biofuels, bioremediation, new bacteria with potential commercial applications, antibiotic discovery, and livestock/plant microbiome interactions.


June 1, 2021  |  

A comprehensive study of the sugar pine (Pinus lambertiana) transcriptome implemented through diverse next-generation sequencing approaches

The assembly, annotation, and characterization of the sugar pine (Pinus lambertiana Dougl.) transcriptome represents an opportunity to study the genetic mechanisms underlying resistance to the invasive white pine blister rust (Cronartium ribicola) as well as responses to other abiotic stresses. The assembled transcripts also provide a resource to improve the genome assembly. We selected a diverse set of tissues allowing the first comprehensive evaluation of the sugar pine gene space. We have combined short read sequencing technologies (Illumina MiSeq and HiSeq) with the relatively new Pacific Biosciences Iso-Seq approach. From the 2.5 billion and 1.6 million Illumina and PacBio (46 SMRT cells) reads, 33,720 unigenes were de novo assembled. Comparison of sequencing technologies revealed improved coverage with Illumina HiSeq reads and better splice variant detection with PacBio Iso-Seq reads. The genes identified as unique to each library ranges from 199 transcripts (basket seedling) to 3,482 transcripts (female cones). In total, 10,026 transcripts were shared by all libraries. Genes differentially expressed in response to these provided insight on abiotic and biotic stress responses. To analyze orthologous sequences, we compared the translated sequences against 19 plant species, identifying 7,229 transcripts that clustered uniquely among the conifers. We have generated here a high quality transcriptome from one WPBR susceptible and one WPBR resistant sugar pine individual. Through the comprehensive tissue sampling and the depth of the sequencing achieved, detailed information on disease resistance can be further examined.


June 1, 2021  |  

Comparative metagenome-assembled genome analysis of “Candidatus Lachnocurva vaginae”, formerly known as Bacterial Vaginosis Associated bacterium – 1 (BVAB1)

Bacterial Vaginosis Associated bacterium 1 (BVAB1) is an as-yet uncultured bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales. As its name suggests, this bacterium is often associated with bacterial vaginosis (BV), a common vaginal disorder that has been shown to increase a woman’s risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae infections as well as preterm birth. Further, BVAB1 is associated with the persistence of BV following metronidazole treatment, increased vaginal inflammation, and adverse obstetrics outcomes. There is no available complete genome sequence of BVAB1, which has made it di?cult to mechanistically understand its role in disease. We present here a circularized metagenome-assembled genome (cMAG) of B VAB1 as well as a comparative analysis including an additional six metagenome-assembled genomes (MAGs) of this species. These sequences were derived from cervicovaginal samples of seven separate women. The cMAG is 1.649 Mb in size and encodes 1,578 genes. We propose to rename BVAB1 to “Candidatus Lachnocurva vaginae” based on phylogenetic analyses, and provide genomic evidence that this candidate species may metabolize D-lactate, produce trimethylamine (one of the chemicals responsible for BV-associated odor), and be motile. The cMAG and the six MAGs are valuable resources that will further contribute to our understanding of the heterogeneous etiology of bacterial vaginosis.


June 1, 2021  |  

Low-input single molecule HiFi sequencing for metagenomic samples

HiFi sequencing on the PacBio Sequel II System enables complete microbial community profiling of complex metagenomic samples using whole genome shotgun sequences. With HiFi sequencing, highly accurate long reads overcome the challenges posed by the presence of intergenic and extragenic repeat elements in microbial genomes, thus greatly improving phylogenetic profiling and sequence assembly. Recent improvements in library construction protocols enable HiFi sequencing starting from as low as 5 ng of input DNA. Here, we demonstrate comparative analyses of a control sample of known composition and a human fecal sample from varying amounts of input genomic DNA (1 ug, 200 ng, 5 ng), and present the corresponding library preparation workflows for standard, low input, and Ultra-Low methods. We demonstrate that the metagenome assembly, taxonomic assignment, and gene finding analyses are comparable across all methods for both samples, providing access to HiFi sequencing even for DNA-limited sample types.


April 21, 2020  |  

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases.

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with ‘ready-to-use’ deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.


April 21, 2020  |  

Tracking short-term changes in the genetic diversity and antimicrobial resistance of OXA-232-producing Klebsiella pneumoniae ST14 in clinical settings.

To track stepwise changes in genetic diversity and antimicrobial resistance in rapidly evolving OXA-232-producing Klebsiella pneumoniae ST14, an emerging carbapenem-resistant high-risk clone, in clinical settings.Twenty-six K. pneumoniae ST14 isolates were collected by the Korean Nationwide Surveillance of Antimicrobial Resistance system over the course of 1 year. Isolates were subjected to whole-genome sequencing and MIC determinations using 33 antibiotics from 14 classes.Single-nucleotide polymorphism (SNP) typing identified 72 unique SNP sites spanning the chromosomes of the isolates, dividing them into three clusters (I, II and III). The initial isolate possessed two plasmids with 18 antibiotic-resistance genes, including blaOXA-232, and exhibited resistance to 11 antibiotic classes. Four other plasmids containing 12 different resistance genes, including blaCTX-M-15 and strA/B, were introduced over time, providing additional resistance to aztreonam and streptomycin. Moreover, chromosomal integration of insertion sequence Ecp1-blaCTX-M-15 mediated the inactivation of mgrB responsible for colistin resistance in four isolates from cluster III. To the best of our knowledge, this is the first description of K. pneumoniae ST14 resistant to both carbapenem and colistin in South Korea. Furthermore, although some acquired genes were lost over time, the retention of 12 resistance genes and inactivation of mgrB provided resistance to 13 classes of antibiotics.We describe stepwise changes in OXA-232-producing K. pneumoniae ST14 in vivo over time in terms of antimicrobial resistance. Our findings contribute to our understanding of the evolution of emerging high-risk K. pneumoniae clones and provide reference data for future outbreaks.Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Analyses of the Complete Genome Sequence of the Strain Bacillus pumilus ZB201701 Isolated from Rhizosphere Soil of Maize under Drought and Salt Stress.

Bacillus pumilus ZB201701 is a rhizobacterium with the potential to promote plant growth and tolerance to drought and salinity stress. We herein present the complete genome sequence of the Gram-positive bacterium B. pumilus ZB201701, which consists of a linear chromosome with 3,640,542 base pairs, 3,608 protein-coding sequences, 24 ribosomal RNAs, and 80 transfer RNAs. Genome analyses using bioinformatics revealed some of the putative gene clusters involved in defense mechanisms. In addition, activity analyses of the strain under salt and simulated drought stress suggested its potential tolerance to abiotic stress. Plant growth-promoting bacteria-based experiments indicated that the strain promotes the salt tolerance of maize. The complete genome of B. pumilus ZB201701 provides valuable insights into rhizobacteria-mediated salt and drought tolerance and rhizobacteria-based solutions for abiotic stress in agriculture.


April 21, 2020  |  

Biochemical characterization of a novel cold-adapted agarotetraose-producing a-agarase, AgaWS5, from Catenovulum sediminis WS1-A.

Although many ß-agarases that hydrolyze the ß-1,4 linkages of agarose have been biochemically characterized, only three a-agarases that hydrolyze the a-1,3 linkages are reported to date. In this study, a new a-agarase, AgaWS5, from Catenovulum sediminis WS1-A, a new agar-degrading marine bacterium, was biochemically characterized. AgaWS5 belongs to the glycoside hydrolase (GH) 96 family. AgaWS5 consists of 1295 amino acids (140 kDa) and has the 65% identity to an a-agarase, AgaA33, obtained from an agar-degrading bacterium Thalassomonas agarivorans JAMB-A33. AgaWS5 showed the maximum activity at a pH and temperature of 8 and 40 °C, respectively. AgaWS5 showed a cold-tolerance, and it retained more than 40% of its maximum enzymatic activity at 10 °C. AgaWS5 is predicted to have several calcium-binding sites. Thus, its activity was slightly enhanced in the presence of Ca2+, and was strongly inhibited by EDTA. The Km and Vmax of AgaWS5 for agarose were 10.6 mg/mL and 714.3 U/mg, respectively. Agarose-liquefication, thin layer chromatography, and mass and NMR spectroscopic analyses demonstrated that AgaWS5 is an endo-type a-agarase that degrades agarose and mainly produces agarotetraose. Thus, in this study, a novel cold-adapted GH96 agarotetraose-producing a-agarase was identified.


April 21, 2020  |  

Complete genome sequence of Marinobacter sp. LQ44, a haloalkaliphilic phenol-degrading bacterium isolated from a deep-sea hydrothermal vent

Marinobacter sp. strain LQ44, an alkaliphile and moderate halophile from a deep-sea hydrothermal vent on the East Pacific Rise, is a novel phenol-degrading bacterium that is capable of utilizing phenol as sole carbon and energy sources. Here, we present the complete genome sequence of strain LQ44, which consists of 4,435,564?bp with a circular chromosome, 4164 protein-coding genes, 3 rRNA operons and 50 tRNAs. Genome analysis revealed that strain LQ44 may degrade phenol via meta-cleavage pathway. The LQ44 genome contains multiple genes involved in pH adaptation and osmotic adjustment. Genes related to hydrocarbon degradation, aerobic denitrification and potential industrial important enzymes were also identified from the genome. To our knowledge, this is the first report of a genome sequence of a haloalkaliphilic phenol-degrading bacterium, which will provide insights into the survival of this bacterium under salt-alkali conditions and the potential for biotechnological applications.


April 21, 2020  |  

Paenibacillus albus sp. nov., a UV radiation-resistant bacterium isolated from soil in Korea.

A novel Gram-stain-positive, motile, white color and endospore-forming bacterium, designated 18JY67-1T, was isolated from soil in Jeju Island, Korea. The strain grow at 15-42 °C (optimum 30 °C) in R2A medium at pH (6.0-9.5) (optimum 7.5). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 18JY67-1T formed a distinct lineage within the family Paenibacillaceae (order Bacillales, class Bacilli), and was closely related to Paenibacillus rhizoryzae (KP675984; 96.9% 16S rRNA gene sequence similarity). The major cellular fatty acids of the strain 18JY67-1T were C16:0 and anteiso-C15:0. The predominant respiratory quinones were MK-7. The major polar lipid was identified as diphosphatidylglycerol. On the basis of phenotypic, chemotaxonomic and genotypic properties clearly indicated that isolate 18JY67-1T represents a novel species within the genus Paenibacillus, for which the name Paenibacillus flavus sp. nov. is proposed. The type strain of Paenibacillus flavus is 18JY67-1T (=?KCTC 33959T =?JCM 33184T).


April 21, 2020  |  

Complete genome sequence of Paracoccus sp. Arc7-R13, a silver nanoparticles synthesizing bacterium isolated from Arctic Ocean sediments

Paracoccus sp. Arc7-R13, a silver nanoparticles (AgNPs) synthesizing bacterium, was isolated from Arctic Ocean sediment. Here we describe the complete genome of Paracoccus sp. Arc7-R13. The complete genome contains 4,040,012?bp with 66.66?mol%?G?+?C content, including one circular chromosome of 3,231,929?bp (67.45?mol%?G?+?C content), and eight plasmids with length ranging from 24,536?bp to 199,685?bp. The genome contains 3835 protein-coding genes (CDSs), 49 tRNA genes, as well as 3 rRNA operons as 16S-23S-5S rRNA. Based on the gene annotation and Swiss-Prot analysis, a total of 15 genes belonging to 11 kinds, including silver exporting P-type ATPase (SilP), alkaline phosphatase, nitroreductase, thioredoxin reductase, NADPH dehydrogenase and glutathione peroxidase, might be related to the synthesis of AgNPs. Meanwhile, many additional genes associated with synthesis of AgNPs such as protein-disulfide isomerase, c-type cytochrome, glutathione synthase and dehydrogenase reductase were also identified.


April 21, 2020  |  

Antibiotic susceptibility of plant-derived lactic acid bacteria conferring health benefits to human.

Lactic acid bacteria (LAB) confer health benefits to human when administered orally. We have recently isolated several species of LAB strains from plant sources, such as fruits, vegetables, flowers, and medicinal plants. Since antibiotics used to treat bacterial infection diseases induce the emergence of drug-resistant bacteria in intestinal microflora, it is important to evaluate the susceptibility of LAB strains to antibiotics to ensure the safety and security of processed foods. The aim of the present study is to determine the minimum inhibitory concentration (MIC) of antibiotics against several plant-derived LAB strains. When aminoglycoside antibiotics, such as streptomycin (SM), kanamycin (KM), and gentamicin (GM), were evaluated using LAB susceptibility test medium (LSM), the MIC was higher than when using Mueller-Hinton (MH) medium. Etest, which is an antibiotic susceptibility assay method consisting of a predefined gradient of antibiotic concentrations on a plastic strip, is used to determine the MIC of antibiotics world-wide. In the present study, we demonstrated that Etest was particularly valuable while testing LAB strains. We also show that the low susceptibility of the plant-derived LAB strains against each antibiotic tested is due to intrinsic resistance and not acquired resistance. This finding is based on the whole-genome sequence information reflecting the horizontal spread of the drug-resistance genes in the LAB strains.


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