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September 22, 2019  |  

Comparative genomics and identification of an enterotoxin-bearing pathogenicity island, SEPI-1/SECI-1, in Staphylococcus epidermidis pathogenic strains.

Staphylococcus epidermidis is a leading cause of nosocomial infections, majorly resistant to beta-lactam antibiotics, and may transfer several mobile genetic elements among the members of its own species, as well as to Staphylococcus aureus; however, a genetic exchange from S. aureus to S. epidermidis remains controversial. We recently identified two pathogenic clinical strains of S. epidermidis that produce a staphylococcal enterotoxin C3-like (SEC) similar to that by S. aureus pathogenicity islands. This study aimed to determine the genetic environment of the SEC-coding sequence and to identify the mobile genetic elements. Whole-genome sequencing and annotation of the S. epidermidis strains were performed using Illumina technology and a bioinformatics pipeline for assembly, which provided evidence that the SEC-coding sequences were located in a composite pathogenicity island that was previously described in the S. epidermidis strain FRI909, called SePI-1/SeCI-1, with 83.8-89.7% nucleotide similarity. Various other plasmids were identified, particularly p_3_95 and p_4_95, which carry antibiotic resistance genes (hsrA and dfrG, respectively), and share homologies with SAP085A and pUSA04-2-SUR11, two plasmids described in S. aureus. Eventually, one complete prophage was identified, FSE90, sharing 30 out of 52 coding sequences with the Acinetobacter phage vB_AbaM_IME200. Thus, the SePI-1/SeCI-1 pathogenicity island was identified in two pathogenic strains of S. epidermidis that produced a SEC enterotoxin causing septic shock. These findings suggest the existence of in vivo genetic exchange from S. aureus to S. epidermidis.


September 22, 2019  |  

Complete genome sequence and analysis of the industrial Saccharomyces cerevisiae strain N85 used in Chinese rice wine production.

Chinese rice wine is a popular traditional alcoholic beverage in China, while its brewing processes have rarely been explored. We herein report the first gapless, near-finished genome sequence of the yeast strain Saccharomyces cerevisiae N85 for Chinese rice wine production. Several assembly methods were used to integrate Pacific Bioscience (PacBio) and Illumina sequencing data to achieve high-quality genome sequencing of the strain. The genome encodes more than 6,000 predicted proteins, and 238 long non-coding RNAs, which are validated by RNA-sequencing data. Moreover, our annotation predicts 171 novel genes that are not present in the reference S288c genome. We also identified 65,902 single nucleotide polymorphisms and small indels, many of which are located within genic regions. Dozens of larger copy-number variations and translocations were detected, mainly enriched in the subtelomeres, suggesting these regions may be related to genomic evolution. This study will serve as a milestone in studying of Chinese rice wine and related beverages in China and in other countries. It will help to develop more scientific and modern fermentation processes of Chinese rice wine, and explore metabolism pathways of desired and harmful components in Chinese rice wine to improve its taste and nutritional value.© The Author(s) 2018. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.


September 22, 2019  |  

Genetic separation of Listeria monocytogenes causing central nervous system infections in animals.

Listeria monocytogenes is a foodborne pathogen that causes abortion, septicemia, gastroenteritis and central nervous system (CNS) infections in ruminants and humans. L. monocytogenes strains mainly belong to two distinct phylogenetic groups, named lineages I and II. In general, clinical cases in humans and animals, in particular CNS infections, are caused by lineage I strains, while most of the environmental and food strains belong to lineage II. Little is known about why lineage I is more virulent than lineage II, even though various molecular factors and mechanisms associated with pathogenesis are known. In this study, we have used a variety of whole genome sequence analyses and comparative genomic tools in order to find characteristics that distinguish lineage I from lineage II strains and CNS infection strains from non-CNS strains. We analyzed 225 strains and identified single nucleotide variants between lineages I and II, as well as differences in the gene content. Using a novel approach based on Reads Per Kilobase per Million Mapped (RPKM), we identified 167 genes predominantly absent in lineage II but present in lineage I. These genes are mostly encoding for membrane-associated proteins. Additionally, we found 77 genes that are largely absent in the non-CNS associated strains, while 39 genes are especially lacking in our defined “non-clinical” group. Based on the RPKM analysis and the metadata linked to the L. monocytogenes strains, we identified 6 genes potentially associated with CNS cases, which include a transcriptional regulator, an ABC transporter and a non-coding RNA. Although there is not a clear separation between pathogenic and non-pathogenic strains based on phylogenetic lineages, the presence of the genes identified in our study reveals potential pathogenesis traits in ruminant L. monocytogenes strains. Ultimately, the differences that we have found in our study will help steer future studies in understanding the virulence mechanisms of the most pathogenic L. monocytogenes strains.


September 22, 2019  |  

Complete genome sequence and genomic characterization of Lactobacillus acidophilus LA1 (11869BP).

Our body has natural defense systems to protect against potentially harmful microbes, including the physical and chemical barriers of the intestinal epithelium (Corfield et al., 2000). The physical barrier of the intestinal epithelium protects the host against pathogenic microbes (Anderson et al., 1993), and the intestinal mucosa coated with mucus excretes pathogens from the intestinal tract (Corfield et al., 2000).


September 22, 2019  |  

Pantoea ananatis genetic diversity analysis reveals limited genomic diversity as well as accessory genes correlated with onion pathogenicity.

Pantoea ananatis is a member of the family Enterobacteriaceae and an enigmatic plant pathogen with a broad host range. Although P. ananatis strains can be aggressive on onion causing foliar necrosis and onion center rot, previous genomic analysis has shown that P. ananatis lacks the primary virulence secretion systems associated with other plant pathogens. We assessed a collection of fifty P. ananatis strains collected from Georgia over three decades to determine genetic factors that correlated with onion pathogenic potential. Previous genetic analysis studies have compared strains isolated from different hosts with varying diseases potential and isolation sources. Strains varied greatly in their pathogenic potential and aggressiveness on different cultivated Allium species like onion, leek, shallot, and chive. Using multi-locus sequence analysis (MLSA) and repetitive extragenic palindrome repeat (rep)-PCR techniques, we did not observe any correlation between onion pathogenic potential and genetic diversity among strains. Whole genome sequencing and pan-genomic analysis of a sub-set of 10 strains aided in the identification of a novel series of genetic regions, likely plasmid borne, and correlating with onion pathogenicity observed on single contigs of the genetic assemblies. We named these loci Onion Virulence Regions (OVR) A-D. The OVR loci contain genes involved in redox regulation as well as pectate lyase and rhamnogalacturonase genes. Previous studies have not identified distinct genetic loci or plasmids correlating with onion foliar pathogenicity or pathogenicity on a single host pathosystem. The lack of focus on a single host system for this phytopathgenic disease necessitates the pan-genomic analysis performed in this study.


September 22, 2019  |  

Probiotic and anti-inflammatory potential of Lactobacillus rhamnosus 4B15 and Lactobacillus gasseri 4M13 isolated from infant feces.

A total of 22 Lactobacillus strains, which were isolated from infant feces were evaluated for their probiotic potential along with resistance to low pH and bile salts. Eight isolates (L. reuteri 3M02 and 3M03, L. gasseri 4M13, 4R22, 5R01, 5R02, and 5R13, and L. rhamnosus 4B15) with high tolerance to acid and bile salts, and ability to adhere to the intestine were screened from 22 strains. Further, functional properties of 8 Lactobacillus strains, such as anti-oxidation, inhibition of a-glucosidase activity, cholesterol-lowering, and anti-inflammation were evaluated. The properties were strain-specific. Particularly, two strains of L. rhamnosus, 4B15 (4B15) and L. gasseri 4M13 (4M13) showed considerably higher anti-oxidation, inhibition of a-glucosidase activity, and cholesterol-lowering, and greater inhibition of nitric oxide production than other strains. Moreover, the two selected strains substantially inhibited the release of inflammatory mediators such as TNF-a, IL-6, IL-1ß, and IL-10 stimulated the treatment of RAW 264.7 macrophages with LPS. In addition, whole genome sequencing and comparative genomic analysis of 4B15 and 4M13 indicated them as novel genomic strains. These results suggested that 4B15 and 4M13 showed the highest probiotic potential and have an impact on immune health by modulating pro-inflammatory cytokines.


September 22, 2019  |  

Culture-facilitated comparative genomics of the facultative symbiont Hamiltonella defensa.

Many insects host facultative, bacterial symbionts that confer conditional fitness benefits to their hosts. Hamiltonella defensa is a common facultative symbiont of aphids that provides protection against parasitoid wasps. Protection levels vary among strains of H. defensa that are also differentially infected by bacteriophages named APSEs. However, little is known about trait variation among strains because only one isolate has been fully sequenced. Generating complete genomes for facultative symbionts is hindered by relatively large genome sizes but low abundances in hosts like aphids that are very small. Here, we took advantage of methods for culturing H. defensa outside of aphids to generate complete genomes and transcriptome data for four strains of H. defensa from the pea aphid Acyrthosiphon pisum. Chosen strains also spanned the breadth of the H. defensa phylogeny and differed in strength of protection conferred against parasitoids. Results indicated that strains shared most genes with roles in nutrient acquisition, metabolism, and essential housekeeping functions. In contrast, the inventory of mobile genetic elements varied substantially, which generated strain specific differences in gene content and genome architecture. In some cases, specific traits correlated with differences in protection against parasitoids, but in others high variation between strains obscured identification of traits with likely roles in defense. Transcriptome data generated continuous distributions to genome assemblies with some genes that were highly expressed and others that were not. Single molecule real-time sequencing further identified differences in DNA methylation patterns and restriction modification systems that provide defense against phage infection.


September 22, 2019  |  

Comparative genomics of the Baltic Sea toxic cyanobacteria Nodularia spumigena UHCC 0039 and its response to varying salinity.

Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spumigena UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.


September 22, 2019  |  

Genomic insights into the Acidobacteria reveal strategies for their success in terrestrial environments.

Members of the phylum Acidobacteria are abundant and ubiquitous across soils. We performed a large-scale comparative genome analysis spanning subdivisions 1, 3, 4, 6, 8 and 23 (n?=?24) with the goal to identify features to help explain their prevalence in soils and understand their ecophysiology. Our analysis revealed that bacteriophage integration events along with transposable and mobile elements influenced the structure and plasticity of these genomes. Low- and high-affinity respiratory oxygen reductases were detected in multiple genomes, suggesting the capacity for growing across different oxygen gradients. Among many genomes, the capacity to use a diverse collection of carbohydrates, as well as inorganic and organic nitrogen sources (such as via extracellular peptidases), was detected – both advantageous traits in environments with fluctuating nutrient environments. We also identified multiple soil acidobacteria with the potential to scavenge atmospheric concentrations of H2 , now encompassing mesophilic soil strains within the subdivision 1 and 3, in addition to a previously identified thermophilic strain in subdivision 4. This large-scale acidobacteria genome analysis reveal traits that provide genomic, physiological and metabolic versatility, presumably allowing flexibility and versatility in the challenging and fluctuating soil environment.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.


September 22, 2019  |  

Two groups of cocirculating, epidemic Clostridiodes difficile strains microdiversify through different mechanisms.

Clostridiodes difficile strains from the NAPCR1/ST54 and NAP1/ST01 types have caused outbreaks despite of their notable differences in genome diversity. By comparing whole genome sequences of 32 NAPCR1/ST54 isolates and 17 NAP1/ST01 recovered from patients infected with C. difficile we assessed whether mutation, homologous recombination (r) or nonhomologous recombination (NHR) through lateral gene transfer (LGT) have differentially shaped the microdiversification of these strains. The average number of single nucleotide polymorphisms (SNPs) in coding sequences (NAPCR1/ST54?=?24; NAP1/ST01?=?19) and SNP densities (NAPCR1/ST54?=?0.54/kb; NAP1/ST01?=?0.46/kb) in the NAPCR1/ST54 and NAP1/ST01 isolates was comparable. However, the NAP1/ST01 isolates showed 3× higher average dN/dS rates (8.35) that the NAPCR1/ST54 isolates (2.62). Regarding r, whereas 31 of the NAPCR1/ST54 isolates showed 1 recombination block (3,301-8,226?bp), the NAP1/ST01 isolates showed no bases in recombination. As to NHR, the pangenome of the NAPCR1/ST54 isolates was larger (4,802 gene clusters, 26% noncore genes) and more heterogeneous (644?±?33 gene content changes) than that of the NAP1/ST01 isolates (3,829 gene clusters, ca. 6% noncore genes, 129?±?37 gene content changes). Nearly 55% of the gene content changes seen among the NAPCR1/ST54 isolates (355?±?31) were traced back to MGEs with putative genes for antimicrobial resistance and virulence factors that were only detected in single isolates or isolate clusters. Congruently, the LGT/SNP rate calculated for the NAPCR1/ST54 isolates (26.8?±?2.8) was 4× higher than the one obtained for the NAP1/ST1 isolates (6.8?±?2.0). We conclude that NHR-LGT has had a greater role in the microdiversification of the NAPCR1/ST54 strains, opposite to the NAP1/ST01 strains, where mutation is known to play a more prominent role.


September 22, 2019  |  

Comparative genomic insights into endofungal lifestyles of two bacterial endosymbionts, Mycoavidus cysteinexigens and Burkholderia rhizoxinica.

Endohyphal bacteria (EHB), dwelling within fungal hyphae, markedly affect the growth and metabolic potential of their hosts. To date, two EHB belonging to the family Burkholderiaceae have been isolated and characterized as new taxa, Burkholderia rhizoxinica (HKI 454T) and Mycoavidus cysteinexigens (B1-EBT), in Japan. Metagenome sequencing was recently reported for Mortierella elongata AG77 together with its endosymbiont M. cysteinexigens (Mc-AG77) from a soil/litter sample in the USA. In the present study, we elucidated the complete genome sequence of B1-EBT and compared it with those of Mc-AG77 and HKI 454T. The genomes of B1-EBT and Mc-AG77 contained a higher level of prophage sequences and were markedly smaller than that of HKI 454T. Although the B1-EBT and Mc-AG77 genomes lacked the chitinolytic enzyme genes responsible for invasion into fungal cells, they contained several predicted toxin-antitoxin systems including an insecticidal toxin complex and PIN domain imposing an addiction-like mechanism essential for endohyphal growth control during host colonization. Despite the different host fungi, the alignment of amino acid sequences showed that the HKI 454T genome consisted of 1,265 (32.6%) and 1,221 (31.5%) orthologous coding sequences (CDSs) with those of B1-EBT and Mc-AG77, respectively. This comparative study of three phylogenetically associated endosymbionts has provided insights into their origin and evolution, and suggests the later bacterial invasion and adaptation of B1-EBT to its host metabolism.


September 22, 2019  |  

Comparative genome analysis reveals a complex population structure of Legionella pneumophila subspecies.

The majority of Legionnaires’ disease (LD) cases are caused by Legionella pneumophila, a genetically heterogeneous species composed of at least 17 serogroups. Previously, it was demonstrated that L. pneumophila consists of three subspecies: pneumophila, fraseri and pascullei. During an LD outbreak investigation in 2012, we detected that representatives of both subspecies fraseri and pascullei colonized the same water system and that the outbreak-causing strain was a new member of the least represented subspecies pascullei. We used partial sequence based typing consensus patterns to mine an international database for additional representatives of fraseri and pascullei subspecies. As a result, we identified 46 sequence types (STs) belonging to subspecies fraseri and two STs belonging to subspecies pascullei. Moreover, a recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters revealed the presence of a fourth L. pneumophila subspecies that we have termed raphaeli. This subspecies consists of 15 STs. Comparative analysis was conducted using the genomes of multiple members of all four L. pneumophila subspecies. Whereas each subspecies forms a distinct phylogenetic clade within the L. pneumophila species, they share more average nucleotide identity with each other than with other Legionella species. Unique genes for each subspecies were identified and could be used for rapid subspecies detection. Improved taxonomic classification of L. pneumophila strains may help identify environmental niches and virulence attributes associated with these genetically distinct subspecies. Published by Elsevier B.V.


September 22, 2019  |  

Horizontal antimicrobial resistance transfer drives epidemics of multiple Shigella species.

Horizontal gene transfer has played a role in developing the global public health crisis of antimicrobial resistance (AMR). However, the dynamics of AMR transfer through bacterial populations and its direct impact on human disease is poorly elucidated. Here, we study parallel epidemic emergences of multiple Shigella species, a priority AMR organism, in men who have sex with men to gain insight into AMR emergence and spread. Using genomic epidemiology, we show that repeated horizontal transfer of a single AMR plasmid among Shigella enhanced existing and facilitated new epidemics. These epidemic patterns contrasted with slighter, slower increases in disease caused by organisms with vertically inherited (chromosomally encoded) AMR. This demonstrates that horizontal transfer of AMR directly affects epidemiological outcomes of globally important AMR pathogens and highlights the need for integration of genomic analyses into all areas of AMR research, surveillance and management.


September 22, 2019  |  

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) ‘Hongyang’ draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models.A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within ‘Hongyang’ The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned ‘Hort16A’ cDNAs and comparing with the predicted protein models for Red5 and both the original ‘Hongyang’ assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised ‘Hongyang’ annotation, respectively, compared with 90.9% to the Red5 models.Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.


September 22, 2019  |  

Genome evolution across 1,011 Saccharomyces cerevisiae isolates.

Large-scale population genomic surveys are essential to explore the phenotypic diversity of natural populations. Here we report the whole-genome sequencing and phenotyping of 1,011 Saccharomyces cerevisiae isolates, which together provide an accurate evolutionary picture of the genomic variants that shape the species-wide phenotypic landscape of this yeast. Genomic analyses support a single ‘out-of-China’ origin for this species, followed by several independent domestication events. Although domesticated isolates exhibit high variation in ploidy, aneuploidy and genome content, genome evolution in wild isolates is mainly driven by the accumulation of single nucleotide polymorphisms. A common feature is the extensive loss of heterozygosity, which represents an essential source of inter-individual variation in this mainly asexual species. Most of the single nucleotide polymorphisms, including experimentally identified functional polymorphisms, are present at very low frequencies. The largest numbers of variants identified by genome-wide association are copy-number changes, which have a greater phenotypic effect than do single nucleotide polymorphisms. This resource will guide future population genomics and genotype-phenotype studies in this classic model system.


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