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September 22, 2019

Whole genome analysis reveals the diversity and evolutionary relationships between necrotic enteritis-causing strains of Clostridium perfringens.

Clostridium perfringens causes a range of diseases in animals and humans including necrotic enteritis in chickens and food poisoning and gas gangrene in humans. Necrotic enteritis is of concern in commercial chicken production due to the cost of the implementation of infection control measures and to productivity losses. This study has focused on the genomic analysis of a range of chicken-derived C. perfringens isolates, from around the world and from different years. The genomes were sequenced and compared with 20 genomes available from public databases, which were from a diverse collection of isolates from chickens, other animals, and humans. We used a distance based phylogeny that was constructed based on gene content rather than sequence identity. Similarity between strains was defined as the number of genes that they have in common divided by their total number of genes. In this type of phylogenetic analysis, evolutionary distance can be interpreted in terms of evolutionary events such as acquisition and loss of genes, whereas the underlying properties (the gene content) can be interpreted in terms of function. We also compared these methods to the sequence-based phylogeny of the core genome.Distinct pathogenic clades of necrotic enteritis-causing C. perfringens were identified. They were characterised by variable regions encoded on the chromosome, with predicted roles in capsule production, adhesion, inhibition of related strains, phage integration, and metabolism. Some strains have almost identical genomes, even though they were isolated from different geographic regions at various times, while other highly distant genomes appear to result in similar outcomes with regard to virulence and pathogenesis.The high level of diversity in chicken isolates suggests there is no reliable factor that defines a chicken strain of C. perfringens, however, disease-causing strains can be defined by the presence of netB-encoding plasmids. This study reveals that horizontal gene transfer appears to play a significant role in genetic variation of the C. perfringens chromosome as well as the plasmid content within strains.

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September 22, 2019

Coordinated regulation of core and accessory genes in the multipartite genome of Sinorhizobium fredii.

Prokaryotes benefit from having accessory genes, but it is unclear how accessory genes can be linked with the core regulatory network when developing adaptations to new niches. Here we determined hierarchical core/accessory subsets in the multipartite pangenome (composed of genes from the chromosome, chromid and plasmids) of the soybean microsymbiont Sinorhizobium fredii by comparing twelve Sinorhizobium genomes. Transcriptomes of two S. fredii strains at mid-log and stationary growth phases and in symbiotic conditions were obtained. The average level of gene expression, variation of expression between different conditions, and gene connectivity within the co-expression network were positively correlated with the gene conservation level from strain-specific accessory genes to genus core. Condition-dependent transcriptomes exhibited adaptive transcriptional changes in pangenome subsets shared by the two strains, while strain-dependent transcriptomes were enriched with accessory genes on the chromid. Proportionally more chromid genes than plasmid genes were co-expressed with chromosomal genes, while plasmid genes had a higher within-replicon connectivity in expression than chromid ones. However, key nitrogen fixation genes on the symbiosis plasmid were characterized by high connectivity in both within- and between-replicon analyses. Among those genes with host-specific upregulation patterns, chromosomal znu and mdt operons, encoding a conserved high-affinity zinc transporter and an accessory multi-drug efflux system, respectively, were experimentally demonstrated to be involved in host-specific symbiotic adaptation. These findings highlight the importance of integrative regulation of hierarchical core/accessory components in the multipartite genome of bacteria during niche adaptation and in shaping the prokaryotic pangenome in the long run.

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September 22, 2019

Convergent loss of ABC transporter genes from Clostridioides difficile genomes is associated with impaired tyrosine uptake and p-cresol production.

We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p-cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile, with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile, we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291.

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September 22, 2019

Isolation, functional characterization and transmissibility of p3PS10, a multidrug resistance plasmid of the fish pathogen Piscirickettsia salmonis.

Antibiotic resistance is a major public health concern due to its association with the loss of efficacy of antimicrobial therapies. Horizontal transfer events may play a significant role in the dissemination of resistant bacterial phenotypes, being mobilizable plasmids a well-known mechanism. In this study, we aimed to gain insights into the genetics underlying the development of antibiotic resistance by Piscirickettsia salmonis isolates, a bacterial fish pathogen and causative agent of salmonid piscirickettsiosis, and the main target of antibiotics used in Chilean salmon farming. We provide experimental evidence that the plasmid p3PS10, which harbors multidrug resistance genes for chloramphenicol (cat2), tetracyclines [tet(31)], aminoglycosides (sat1 and aadA1), and sulfonamides (sul2), is carried by a group of P. salmonis isolates exhibiting a markedly reduced susceptibility to oxytetracycline in vitro (128-256 µg/mL of minimal inhibitory concentration, MIC). Antibiotic susceptibility analysis extended to those antibiotics showed that MIC of chloramphenicol, streptomycin, and sulfamethoxazole/trimethoprim were high, but the MIC of florfenicol remained at the wild-type level. By means of molecular cloning, we demonstrate that those genes encoding putative resistance markers are indeed functional. Interestingly, mating assays clearly show that p3PS10 is able to be transferred into and replicate in different hosts, thereby conferring phenotypes similar to those found in the original host. According to epidemiological data, this strain is distributed across aquaculture settings in southern Chile and is likely to be responsible for oxytetracycline treatment failures. This work demonstrates that P. salmonis is more versatile than it was thought, capable of horizontally transferring DNA, and probably playing a role as a vector of resistance traits among the seawater bacterial population. However, the low transmission frequency of p3PS10 suggests a negligible chance of resistance markers being spread to human pathogens.

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September 22, 2019

Isolation and characterization of Bacillus sp. GFP-2, a novel Bacillus strain with antimicrobial activities, from Whitespotted bamboo shark intestine.

The abuse of antibiotics and following rapidly increasing of antibiotic-resistant pathogens is the serious threat to our society. Natural products from microorganism are regarded as the important substitution antimicrobial agents of antibiotics. We isolated a new strain, Bacillus sp. GFP-2, from the Chiloscyllium plagiosum (Whitespotted bamboo shark) intestine, which showed great inhibitory effects on the growth of both Gram-positive and Gram-negative bacteria. Additionally, the growth of salmon was effectively promoted when fed with inactivated strain GFP-2 as the inhibition agent of pathogenic bacteria. The genes encoding antimicrobial peptides like LCI, YFGAP and hGAPDH and gene clusters for secondary metabolites and bacteriocins, such as difficidin, bacillibactin, bacilysin, surfactin, butirosin, macrolactin, bacillaene, fengycin, lanthipeptides and LCI, were predicted in the genome of Bacillus sp. GFP-2, which might be expressed and contribute to the antimicrobial activities of this strain. The gene encoding ß-1,3-1,4-glucanase was successfully cloned from the genome and this protein was detected in the culture supernatant of Bacillus sp. GFP-2 by the antibody produced in rabbit immunized with the recombinant ß-1,3-1,4-glucanase, indicating that this strain could express ß-1,3-1,4-glucanase, which might partially contribute to its antimicrobial activities. This study can enhance a better understanding of the mechanism of antimicrobial activities in genus Bacillus and provide a useful material for the biotechnology study in antimicrobial agent development.

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September 22, 2019

Genomic and probiotic characterization of SJP-SNU strain of Pichia kudriavzevii.

The yeast strain SJP-SNU was investigated as a probiotic and was characterized with respect to growth temperature, bile salt resistance, hydrogen sulfide reducing activity, intestinal survival ability and chicken embryo pathogenicity. In addition, we determined the complete genomic and mitochondrial sequences of SJP-SNU and conducted comparative genomics analyses. SJP-SNU grew rapidly at 37 °C and formed colonies on MacConkey agar containing bile salt. SJP-SNU reduced hydrogen sulfide produced by Salmonella serotype Enteritidis and, after being fed to 4-week-old chickens, could be isolated from cecal feces. SJP-SNU did not cause mortality in 10-day-old chicken embryos. From 13 initial contigs, 11 were finally assembled and represented 10 chromosomal sequences and 1 mitochondrial DNA sequence. Comparative genomic analyses revealed that SJP-SNU was a strain of Pichia kudriavzevii. Although SJP-SNU possesses pathogenicity-related genes, they showed very low amino acid sequence identities to those of Candida albicans. Furthermore, SJP-SNU possessed useful genes, such as phytases and cellulase. Thus, SJP-SNU is a useful yeast possessing the basic traits of a probiotic, and further studies to demonstrate its efficacy as a probiotic in the future may be warranted.

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September 22, 2019

Biosynthesis of the 15-membered ring depsipeptide neoantimycin.

Antimycins are a family of natural products possessing outstanding biological activities and unique structures, which have intrigued chemists for over a half century. Of particular interest are the ring-expanded antimycins that show promising anticancer potential and whose biosynthesis remains uncharacterized. Specifically, neoantimycin and its analogs have been shown to be effective regulators of the oncogenic proteins GRP78/BiP and K-Ras. The neoantimycin structural skeleton is built on a 15-membered tetralactone ring containing one methyl, one hydroxy, one benzyl, and three alkyl moieties, as well as an amide linkage to a conserved 3-formamidosalicylic acid moiety. Although the biosynthetic gene cluster for neoantimycins was recently identified, the enzymatic logic that governs the synthesis of neoantimycins has not yet been revealed. In this work, the neoantimycin gene cluster is identified, and an updated sequence and annotation is provided delineating a nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) hybrid scaffold. Using cosmid expression and CRISPR/Cas-based genome editing, several heterologous expression strains for neoantimycin production are constructed in two separate Streptomyces species. A combination of in vivo and in vitro analysis is further used to completely characterize the biosynthesis of neoantimycins including the megasynthases and trans-acting domains. This work establishes a set of highly tractable hosts for producing and engineering neoantimycins and their C11 oxidized analogs, paving the way for neoantimycin-based drug discovery and development.

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September 22, 2019

Nucleotide-binding resistance gene signatures in sugar beet, insights from a new reference genome.

Nucleotide-binding (NB-ARC), leucine-rich-repeat genes (NLRs) account for 60.8% of resistance (R) genes molecularly characterized from plants. NLRs exist as large gene families prone to tandem duplication and transposition, with high sequence diversity among crops and their wild relatives. This diversity can be a source of new disease resistance, but difficulty in distinguishing specific sequences from homologous gene family members hinders characterization of resistance for improving crop varieties. Current genome sequencing and assembly technologies, especially those using long-read sequencing, are improving resolution of repeat-rich genomic regions and clarifying locations of duplicated genes, such as NLRs. Using the conserved NB-ARC domain as a model, 231 tentative NB-ARC loci were identified in a highly contiguous genome assembly of sugar beet, revealing diverged and truncated NB-ARC signatures as well as full-length sequences. The NB-ARC-associated proteins contained NLR resistance gene domains, including TIR, CC, and LRR, as well as other integrated domains. Phylogenetic relationships of partial and complete domains were determined, and patterns of physical clustering in the genome were evaluated. Comparison of sugar beet NB-ARC domains to validated R genes from monocots and eudicots suggested extensive B. vulgaris-specific subfamily expansions. The NLR landscape in the rhizomania resistance conferring Rz region of Chromosome 3 was characterized, identifying 26 NLR-like sequences spanning 20 MB. This work presents the first detailed view of NLR family composition in a member of the Caryophyllales, builds a foundation for additional disease resistance work in B. vulgaris, and demonstrates an additional nucleic-acid-based method for NLR prediction in non-model plant species. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.

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September 22, 2019

Characterization of the complete sequences and stability of plasmids carrying the genes aac(6′)-Ib-cr or qnrS in Shigella flexneri in the Hangzhou area of China.

The aim of this study was to explore the fluoroquinolone resistance mechanism of aac (6′)-Ib-cr and qnrS gene by comparing complete sequences and stability of the aac(6′)-Ib-cr- and qnrS-positive plasmids from Shigella isolates in the Hangzhou area of China. The complete sequences of four newly acquired plasmids carrying aac(6′)-Ib-cr or qnrS were compared with those of two plasmids obtained previously and two similar reference Escherichia coli plasmids. The results showed that the length, antibiotic resistance genes and genetic environment were different among the plasmids. Moreover, the plasmid stability of three wild-type isolates and five plasmid transformants carrying aac(6′)-Ib-cr and/or qnrS was measured in vitro, and all eight isolates were found to have lost their aac(6′)-Ib-cr- or qnrS-positive plasmids to a different extent at different stages. When the plasmids were electroporated into Shigella flexneri or they lost positive plasmids, the MICs of ciprofloxacin increased or decreased two- to eightfold for aac(6′)-Ib-cr-positive plasmids and 16- to 32-fold for qnrS-positive plasmids. To our knowledge, this is the first report comparing the complete sequences and describing stability for the aac(6′)-Ib-cr- and qnrS-positive plasmids from Shigella isolates.

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September 22, 2019

Comparative genomics analysis of plasmid pPV989-94 from a clinical isolate of Pantoea vagans PV989.

Pantoea vagans, a gram-negative bacterium from the genus Pantoea and family Enterobacteriaceae, is present in various natural environments and considered to be plant endophytes. We isolated the Pantoea vagans PV989 strain from the clinic and sequenced its whole genome. Besides a chromosome DNA molecule, it also harboured three large plasmids. A comparative genomics analysis was performed for the smallest plasmid, pPV989-94. It can be divided into four regions, including three conservative regions related to replication (R1), transfer conjugation (R2), and transfer leading (R3), and one variable region (R4). Further analysis showed that pPV989-94 is most similar to plasmids LA637P2 and pEA68 of Erwinia amylovora strains isolated from fruit trees. These three plasmids share three conservative regions (R1, R2, and R3). Interestingly, a fragment (R4′) in R4, mediated by phage integrase and phage integrase family site-specific recombinase and encoding 9 genes related to glycometabolism, resistance, and DNA repair, was unique in pPV989-94. Homologues of R4′ were found in other plasmids or chromosomes, suggesting that horizontal gene transfer (HGT) occurred among different bacteria of various species or genera. The acquired functional genes may play important roles in the adaptation of bacteria to different hosts or environmental conditions.

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September 22, 2019

Enrichment of the antibiotic resistance gene tet(L) in an alkaline soil fertilized with plant derived organic manure.

Fifteen antibiotic resistance genes (ARGs) and intI1, a gene involved in horizontal gene transfer (HGT) of ARGs, were quantified in three different soil samples from a 22 year old field experiment that had received inorganic fertilizer (NPK), organic manure (OM; a mixture of wheat straw, soybean oil cake and cotton cake), and control fields that had received no fertilizer and manure (CK). Tet(L) was the most abundant ARG in OM, which also contained considerable levels of intI1. Molecular analysis of yearly collected archived soils over the past 22 years showed that tet(L) and intI1 were higher in OM soils than in NPK soils. The relative abundance of tet(L) was essentially constant during these years, while the level of intI1 in OM soils decreased over time. The main genotype of tet(L) was the same in archived and in fresh soil, OM, and irrigation water. Phylogenetic analysis of the 16S rRNA genes of tetracycline-resistant bacteria (TRB) isolates indicated that the Firmucutes carrying tet(L) in OM were similar to those in the OM soil, suggesting that OM transferred TRB into the OM soils where they survived. Almost all of the TRB isolated from OM carried tet(L) and belonged to the Firmicutes. Survival of bacteria from the organic manure that carried tet(L) may be the cause of the increased level of tet(L) in OM soil.

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September 22, 2019

Enrichment of low-density symbiont DNA from minute insects.

Symbioses between bacteria and insects are often associated with changes in important biological traits that can significantly affect host fitness. To a large extent, studies of these interactions have been based on physiological changes or induced phenotypes in the host, and the genetic mechanisms by which symbionts interact with their hosts have only recently become better understood. Learning about symbionts has been challenging in part due to difficulties such as obtaining enough high quality genomic material for high throughput sequencing technology, especially for symbionts present in low titers, and in small or difficult to rear non-model hosts. Here we introduce a new method that substantially increases the yield of bacterial DNA in minute arthropod hosts, and requires less starting material relative to previous published methods. Copyright © 2018 Elsevier B.V. All rights reserved.

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September 22, 2019

Genus-wide assessment of lignocellulose utilization in the extremely thermophilic Caldicellulosiruptor by genomic, pan-genomic and metagenomic analysis

Metagenomic data from Obsidian Pool (Yellowstone National Park, USA) and 13 genome sequences were used to reassess genus-wide biodiversity for the extremely thermophilic Caldicellulosiruptor The updated core genome contains 1,401 ortholog groups (average genome size for 13 species = 2,516 genes). The pangenome, which remains open with a revised total of 3,493 ortholog groups, encodes a variety of multidomain glycoside hydrolases (GHs). These include three cellulases with GH48 domains that are colocated in the glucan degradation locus (GDL) and are specific determinants for microcrystalline cellulose utilization. Three recently sequenced species, Caldicellulosiruptor sp. strain Rt8.B8 (renamed here Caldicellulosiruptor morganii), Thermoanaerobacter cellulolyticus strain NA10 (renamed here Caldicellulosiruptor naganoensis), and Caldicellulosiruptor sp. strain Wai35.B1 (renamed here Caldicellulosiruptor danielii), degraded Avicel and lignocellulose (switchgrass). C. morganii was more efficient than Caldicellulosiruptor bescii in this regard and differed from the other 12 species examined, both based on genome content and organization and in the specific domain features of conserved GHs. Metagenomic analysis of lignocellulose-enriched samples from Obsidian Pool revealed limited new information on genus biodiversity. Enrichments yielded genomic signatures closely related to that of Caldicellulosiruptor obsidiansis, but there was also evidence for other thermophilic fermentative anaerobes (Caldanaerobacter, Fervidobacterium, Caloramator, and Clostridium). One enrichment, containing 89.8% Caldicellulosiruptor and 9.7% Caloramator, had a capacity for switchgrass solubilization comparable to that of C. bescii These results refine the known biodiversity of Caldicellulosiruptor and indicate that microcrystalline cellulose degradation at temperatures above 70°C, based on current information, is limited to certain members of this genus that produce GH48 domain-containing enzymes.IMPORTANCE The genus Caldicellulosiruptor contains the most thermophilic bacteria capable of lignocellulose deconstruction, which are promising candidates for consolidated bioprocessing for the production of biofuels and bio-based chemicals. The focus here is on the extant capability of this genus for plant biomass degradation and the extent to which this can be inferred from the core and pangenomes, based on analysis of 13 species and metagenomic sequence information from environmental samples. Key to microcrystalline hydrolysis is the content of the glucan degradation locus (GDL), a set of genes encoding glycoside hydrolases (GHs), several of which have GH48 and family 3 carbohydrate binding module domains, that function as primary cellulases. Resolving the relationship between the GDL and lignocellulose degradation will inform efforts to identify more prolific members of the genus and to develop metabolic engineering strategies to improve this characteristic. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Comparative genomic analysis of Geosporobacter ferrireducens and its versatility of anaerobic energy metabolism.

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H+ and Na+ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na+-dependent F-type ATPase and H+-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.

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September 22, 2019

Evolutionary history of bacteriophages in the genus Paraburkholderia.

The genus Paraburkholderia encompasses mostly environmental isolates with diverse predicted lifestyles. Genome analyses have shown that bacteriophages form a considerable portion of some Paraburkholderia genomes. Here, we analyzed the evolutionary history of prophages across all Paraburkholderia spp. Specifically, we investigated to what extent the presence of prophages and their distribution affect the diversity/diversification of Paraburkholderia spp., as well as to what extent phages coevolved with their respective hosts. Particular attention was given to the presence of CRISPR-Cas arrays as a reflection of past interactions with phages. We thus analyzed 36 genomes of Paraburkholderia spp., including those of 11 new strains, next to those of three Burkholderia species. Most genomes were found to contain at least one full prophage sequence. The highest number was found in Paraburkholderia sp. strain MF2-27; the nine prophages found amount to up to 4% of its genome. Among all prophages, potential moron genes (e.g., DNA adenine methylase) were found that might be advantageous for host cell fitness. Co-phylogenetic analyses indicated the existence of complex evolutionary scenarios between the different Paraburkholderia hosts and their prophages, including short-term co-speciation, duplication, host-switching and phage loss events. Analysis of the CRISPR-Cas systems showed a record of diverse, potentially recent, phage infections. We conclude that, overall, different phages have interacted in diverse ways with their Paraburkholderia hosts over evolutionary time.

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