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  |  

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av. © 2019 John Wiley & Sons Ltd.


April 21, 2020  |  

Complete genome sequence of Paenisporosarcina antarctica CGMCC 1.6503 T, a marine psychrophilic bacterium isolated from Antarctica

A marine psychrophilic bacterium _Paenisporosarcina antarctica_ CGMCC 1.6503T (= JCM 14646T) was isolated off King George Island, Antarctica (62°13’31? S 58°57’08? W). In this study, we report the complete genome sequence of _Paenisporosarcina antarctica_, which is comprised of 3,972,524?bp with a mean G?+?C content of 37.0%. By gene function and metabolic pathway analyses, studies showed that strain CGMCC 1.6503T encodes a series of genes related to cold adaptation, including encoding fatty acid desaturases, dioxygenases, antifreeze proteins and cold shock proteins, and possesses several two-component regulatory systems, which could assist this strain in responding to the cold stress, the oxygen stress and the osmotic stress in Antarctica. The complete genome sequence of _P. antarctica_ may provide further insights into the genetic mechanism of cold adaptation for Antarctic marine bacteria.


April 21, 2020  |  

Complete genome sequence provides insights into the quorum sensing-related spoilage potential of Shewanella baltica 128 isolated from spoiled shrimp.

Shewanella baltica 128 is a specific spoilage organism (SSO) isolated from the refrigerated shrimp that results in shrimp spoilage. This study reported the complete genome sequencing of this strain, with the primary annotations associated with amino acid transport and metabolism (8.66%), indicating that S. baltica 128 has good potential for degrading proteins. In vitro experiments revealed Shewanella baltica 128 could adapt to the stress conditions by regulating its growth and biofilm formation. Genes that related to the spoilage-related metabolic pathways, including trimethylamine metabolism (torT), sulfur metabolism (cysM), putrescine metabolism (speC), biofilm formation (rpoS) and serine protease production (degS), were identified. Genes (LuxS, pfs, LuxR and qseC) that related to the specific QS system were also identified. Complete genome sequence of S. baltica 128 provide insights into the QS-related spoilage potential, which might provide novel information for the development of new approaches for spoilage detection and prevention based on QS target.Copyright © 2019. Published by Elsevier Inc.


April 21, 2020  |  

Complete genome sequence of Pseudoalteromonas sp. MEBiC 03485, isolated from deep-sea sediment

Pseudoalteromonas strains are widely distributed in the marine environment and most have attracted considerable interest owing to their ability to synthesize biologically active metabolites. In this study, we report and describe the genome sequence of Pseudoalteromonas sp. MEBiC 03485, isolated from the deep-sea sediment of Pacific Ocean at a depth of 2000?m. The complete genome consisted of three contigs with a total genome size of 4,167,407?bp and a GC content of 40.76?l%, and was predicted to contain 4194 protein-coding genes and 131 non-coding RNA genes. The strain MEBiC 03485 genome was also shown to contain genes for diverse metabolic pathways. Genome analysis revealed that the genome of strain MEBiC 03485 was enriched with genes involved in signal transduction, mobile elements, and cold-adaptation, some of which might improve ecological fitness in the deep-sea environment. These findings improve our understanding of microbial adaptation strategies in deep-sea environments.


April 21, 2020  |  

Complete genome sequence of Bacillus velezensis JT3-1, a microbial germicide isolated from yak feces

Bacillus velezensis JT3-1 is a probiotic strain isolated from feces of the domestic yak (Bos grunniens) in the Gansu province of China. It has strong antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, Mannheimia haemolytica, Staphylococcus hominis, Clostridium perfringens, and Mycoplasma bovis. These properties have made the JT3-1 strain the focus of commercial interest. In this study, we describe the complete genome sequence of JT3-1, with a genome size of 3,929,799 bp, 3761 encoded genes and an average GC content of 46.50%. Whole genome sequencing of Bacillus velezensis JT3-1 will lay a good foundation for elucidation of the mechanisms of its antimicrobial activity, and for its future application.


April 21, 2020  |  

Complete genome screening of clinical MRSA isolates identifies lineage diversity and provides full resolution of transmission and outbreak events

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices, but most applications are focused on conserved core genomic regions due to limitations of short-read technologies. In this study we established a long-read technology-based WGS screening program of all first-episode MRSA blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads revealed widespread gain/loss of accessory mobile genetic elements among established hospital- and community-associated lineages impacting >10% of each genome, and frequent megabase-scale inversions between endogenous prophages. We also characterized an outbreak of a CC5/ST105/USA100 clone among 3 adults and 18 infants in a neonatal intensive care unit (NICU) lasting 7 months. The pattern of changes among complete outbreak genomes provided full spatiotemporal resolution of its origins and progression, which was characterized by multiple sub-transmissions and likely precipitated by equipment sharing. Compared to other hospital strains, the outbreak strain carried distinct mutations and accessory genetic elements that impacted genes with roles in metabolism, resistance and persistence. This included a DNA-recognition domain recombination in the hsdS gene of a Type-I restriction-modification system that altered DNA methylation. RNA-Seq profiling showed that the (epi)genetic changes in the outbreak clone attenuated agr gene expression and upregulated genes involved in stress response and biofilm formation. Overall our findings demonstrate that long-read sequencing substantially improves our ability to characterize accessory genomic elements that impact MRSA virulence and persistence, and provides valuable information for infection control efforts.


April 21, 2020  |  

Genome rearrangements induce biofilm formation in Escherichia coli C, an old model organism with a new application in biofilm research

Escherichia coli C forms more robust biofilms than the other laboratory strains. Biofilm formation and cell aggregation under a high shear force depends on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for LPS synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the -35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene, which may drive its overexpression in biofilm. And finally, E. coli C encodes an additional sigma70 subunit overexpressed in biofilm and driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions and total expression profiles using RNA-seq analyses comparing planktonic and biofilm envirovars provided insights into understanding this regulatory pathway in E. coli.


April 21, 2020  |  

Evolution and global transmission of a multidrug-resistant, community-associated MRSA lineage from the Indian subcontinent

The evolution and global transmission of antimicrobial resistance has been well documented in Gram-negative bacteria and healthcare-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. Here, we trace the recent origins and global spread of a multidrug resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data shows that the clone emerged on the Indian subcontinent in the early 1970s and disseminated rapidly in the 1990s. Short-term outbreaks in community and healthcare settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the divergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional healthcare-associated clones with the epidemiological transmission of community-associated MRSA. Our study demonstrates the importance of whole genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.Importance The Bengal Bay clone (ST772) is a community-acquired and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we show that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally resulting in small-scale community and healthcare outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug-resistance of healthcare-associated S. aureus lineages. This study demonstrates the importance of whole genome sequencing for the surveillance of highly antibiotic resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.


April 21, 2020  |  

Complete Genome Sequence of Desulfovibrio desulfuricans IC1, a Sulfonate-Respiring Anaerobe.

We report the complete genome sequence of the anaerobic, sulfonate-respiring, sulfate-reducing bacterium Desulfovibrio desulfuricans IC1. The genome was assembled into a single 3.25-Mb circular chromosome with 2,680 protein-coding genes identified. Sequencing of sulfonate-metabolizing anaerobes is key for understanding sulfonate degradation and its role in the sulfur cycle.Copyright © 2019 Day et al.


April 21, 2020  |  

Dual Role of gnaA in Antibiotic Resistance and Virulence in Acinetobacter baumannii.

Acinetobacter baumannii is an important Gram-negative pathogen in hospital-related infections. However, treatment options for A. baumannii infections have become limited due to multidrug resistance. Bacterial virulence is often associated with capsule genes found in the K locus, many of which are essential for biosynthesis of the bacterial envelope. However, the roles of other genes in the K locus remain largely unknown. From an in vitro evolution experiment, we obtained an isolate of the virulent and multidrug-resistant A. baumannii strain MDR-ZJ06, called MDR-ZJ06M, which has an insertion by the ISAba16 transposon in gnaA (encoding UDP-N-acetylglucosamine C-6 dehydrogenase), a gene found in the K locus. The isolate showed an increased resistance toward tigecycline, whereas the MIC decreased in the case of carbapenems, cephalosporins, colistin, and minocycline. By using knockout and complementation experiments, we demonstrated that gnaA is important for the synthesis of lipooligosaccharide and capsular polysaccharide and that disruption of the gene affects the morphology, drug susceptibility, and virulence of the pathogen.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Streptococcus oralis subsp. dentisani Produces Monolateral Serine-Rich Repeat Protein Fibrils, One of Which Contributes to Saliva Binding via Sialic Acid.

Our studies reveal that the oral colonizer and cause of infective endocarditis Streptococcus oralis subsp. dentisani displays a striking monolateral distribution of surface fibrils. Furthermore, our data suggest that these fibrils impact the structure of adherent bacterial chains. Mutagenesis studies indicate that these fibrils are dependent on three serine-rich repeat proteins (SRRPs), here named fibril-associated protein A (FapA), FapB, and FapC, and that each SRRP forms a different fibril with a distinct distribution. SRRPs are a family of bacterial adhesins that have diverse roles in adhesion and that can bind to different receptors through modular nonrepeat region domains. Amino acid sequence and predicted structural similarity searches using the nonrepeat regions suggested that FapA may contribute to interspecies interactions, that FapA and FapB may contribute to intraspecies interactions, and that FapC may contribute to sialic acid binding. We demonstrate that a fapC mutant was significantly reduced in binding to saliva. We confirmed a role for FapC in sialic acid binding by demonstrating that the parental strain was significantly reduced in adhesion upon addition of a recombinantly expressed, sialic acid-specific, carbohydrate binding module, while the fapC mutant was not reduced. However, mutation of a residue previously shown to be essential for sialic acid binding did not decrease bacterial adhesion, leaving the precise mechanism of FapC-mediated adhesion to sialic acid to be defined. We also demonstrate that the presence of any one of the SRRPs is sufficient for efficient biofilm formation. Similar structures were observed on all infective endocarditis isolates examined, suggesting that this distribution is a conserved feature of this S. oralis subspecies.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81?kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR The length of the target site was 52 or 55?bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Genomic and Functional Analysis of Emerging Virulent and Multidrug-Resistant Escherichia coli Lineage Sequence Type 648.

The pathogenic extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli lineage ST648 is increasingly reported from multiple origins. Our study of a large and global ST648 collection from various hosts (87 whole-genome sequences) combining core and accessory genomics with functional analyses and in vivo experiments suggests that ST648 is a nascent and generalist lineage, lacking clear phylogeographic and host association signals. By including large numbers of ST131 (n?=?107) and ST10 (n?=?96) strains for comparative genomics and phenotypic analysis, we demonstrate that the combination of multidrug resistance and high-level virulence are the hallmarks of ST648, similar to international high-risk clonal lineage ST131. Specifically, our in silico, in vitro, and in vivo results demonstrate that ST648 is well equipped with biofilm-associated features, while ST131 shows sophisticated signatures indicative of adaption to urinary tract infection, potentially conveying individual ecological niche adaptation. In addition, we used a recently developed NFDS (negative frequency-dependent selection) population model suggesting that ST648 will increase significantly in frequency as a cause of bacteremia within the next few years. Also, ESBL plasmids impacting biofilm formation aided in shaping and maintaining ST648 strains to successfully emerge worldwide across different ecologies. Our study contributes to understanding what factors drive the evolution and spread of emerging international high-risk clonal lineages.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Identification of Initial Colonizing Bacteria in Dental Plaques from Young Adults Using Full-Length 16S rRNA Gene Sequencing.

Development of dental plaque begins with the adhesion of salivary bacteria to the acquired pellicle covering the tooth surface. In this study, we collected in vivo dental plaque formed on hydroxyapatite disks for 6 h from 74 young adults and identified initial colonizing taxa based on full-length 16S rRNA gene sequences. A long-read, single-molecule sequencer, PacBio Sequel, provided 100,109 high-quality full-length 16S rRNA gene sequence reads from the early plaque microbiota, which were assigned to 90 oral bacterial taxa. The microbiota obtained from every individual mostly comprised the 21 predominant taxa with the maximum relative abundance of over 10% (95.8?±?6.2%, mean ± SD), which included Streptococcus species as well as nonstreptococcal species. A hierarchical cluster analysis of their relative abundance distribution suggested three major patterns of microbiota compositions: a Streptococcus mitis/Streptococcus sp. HMT-423-dominant profile, a Neisseria sicca/Neisseria flava/Neisseria mucosa-dominant profile, and a complex profile with high diversity. No notable variations in the community structures were associated with the dental caries status, although the total bacterial amounts were larger in the subjects with a high number of caries-experienced teeth (=8) than in those with no or a low number of caries-experienced teeth. Our results revealed the bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults.IMPORTANCE Selective attachment of salivary bacteria to the tooth surface is an initial and repetitive phase in dental plaque development. We employed full-length 16S rRNA gene sequence analysis with a high taxonomic resolution using a third-generation sequencer, PacBio Sequel, to determine the bacterial composition during early plaque formation in 74 young adults accurately and in detail. The results revealed 21 bacterial taxa primarily involved in early plaque formation on hydroxyapatite disks in young adults, which include several streptococcal species as well as nonstreptococcal species, such as Neisseria sicca/Nflava/Nmucosa and Rothia dentocariosa Given that no notable variations in the microbiota composition were associated with the dental caries status, the maturation process, rather than the specific bacterial species that are the initial colonizers, is likely to play an important role in the development of dysbiotic microbiota associated with dental caries. Copyright © 2019 Ihara et al.


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