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September 22, 2019

Characterization and genomic analyses of Pseudomonas aeruginosa podovirus TC6: establishment of genus Pa11virus.

Phages have attracted a renewed interest as alternative to chemical antibiotics. Although the number of phages is 10-fold higher than that of bacteria, the number of genomically characterized phages is far less than that of bacteria. In this study, phage TC6, a novel lytic virus of Pseudomonas aeruginosa, was isolated and characterized. TC6 consists of an icosahedral head with a diameter of approximately 54 nm and a short tail with a length of about 17 nm, which are characteristics of the family Podoviridae. TC6 can lyse 86 out of 233 clinically isolated P. aeruginosa strains, thus showing application potentials for phage therapy. The linear double-stranded genomic DNA of TC6 consisted of 49796 base pairs and was predicted to contain 71 protein-coding genes. A total of 11 TC6 structural proteins were identified by mass spectrometry. Comparative analysis revealed that the P. aeruginosa phages TC6, O4, PA11, and IME180 shared high similarity at DNA sequence and proteome levels, among which PA11 was the first phage discovered and published. Meanwhile, these phages contain 54 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore proposed that these four phages can be classified as Pa11virus, comprising a new phage genus of Podoviridae that infects Pseudomonas spp. The results of this work promoted our understanding of phage biology, classification, and diversity.

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September 22, 2019

Therapeutic potential of a new jumbo phage that infects Vibrio coralliilyticus, a widespread coral pathogen.

Biological control using bacteriophages is a promising approach for mitigating the devastating effects of coral diseases. Several phages that infect Vibrio coralliilyticus, a widespread coral pathogen, have been isolated, suggesting that this bacterium is permissive to viral infection and is, therefore, a suitable candidate for treatment by phage therapy. In this study, we combined functional and genomic approaches to evaluate the therapeutic potential of BONAISHI, a novel V. coralliilyticus phage, which was isolated from the coral reef in Van Phong Bay (Vietnam). BONAISHI appears to be strictly lytic for several pathogenic strains of V. coralliilyticus and remains infectious over a broad range of environmental conditions. This candidate has an unusually large dsDNA genome (303 kb), with no genes that encode known toxins or implicated in lysogeny control. We identified several proteins involved in host lysis, which may offer an interesting alternative to the use of whole bacteriophages for controlling V. coralliilyticus. A preliminary therapy test showed that adding BONAISHI to an infected culture of Symbiodinium sp. cells reduced the impact of V. coralliilyticus on Symbiodinium sp. photosynthetic activity. This study showed that BONAISHI is able to mitigate V. coralliilyticus infections, making it a good candidate for phage therapy for coral disease.

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September 22, 2019

pYR4 from a Norwegian isolate of Yersinia ruckeri is a putative virulence plasmid encoding both a type IV pilus and a type IV secretion system

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.

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September 22, 2019

Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156).

Exosialidases are glycoside hydrolases that remove a single terminal sialic acid residue from oligosaccharides. They are widely distributed in biology, having been found in prokaryotes, eukaryotes, and certain viruses. Most characterized prokaryotic sialidases are from organisms that are pathogenic or commensal with mammals. However, in this study, we used functional metagenomic screening to seek microbial sialidases encoded by environmental DNA isolated from an extreme ecological niche, a thermal spring. Using recombinant expression of potential exosialidase candidates and a fluorogenic sialidase substrate, we discovered an exosialidase having no homology to known sialidases. Phylogenetic analysis indicated that this protein is a member of a small family of bacterial proteins of previously unknown function. Proton NMR revealed that this enzyme functions via an inverting catalytic mechanism, a biochemical property that is distinct from those of known exosialidases. This unique inverting exosialidase defines a new CAZy glycoside hydrolase family we have designated GH156.© 2018 Chuzel et al.

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September 22, 2019

Alpha- and beta-mannan utilization by marine Bacteroidetes.

Marine microscopic algae carry out about half of the global carbon dioxide fixation into organic matter. They provide organic substrates for marine microbes such as members of the Bacteroidetes that degrade algal polysaccharides using carbohydrate-active enzymes (CAZymes). In Bacteroidetes genomes CAZyme encoding genes are mostly grouped in distinct regions termed polysaccharide utilization loci (PULs). While some studies have shown involvement of PULs in the degradation of algal polysaccharides, the specific substrates are for the most part still unknown. We investigated four marine Bacteroidetes isolated from the southern North Sea that harbour putative mannan-specific PULs. These PULs are similarly organized as PULs in human gut Bacteroides that digest a- and ß-mannans from yeasts and plants respectively. Using proteomics and defined growth experiments with polysaccharides as sole carbon sources we could show that the investigated marine Bacteroidetes express the predicted functional proteins required for a- and ß-mannan degradation. Our data suggest that algal mannans play an as yet unknown important role in the marine carbon cycle, and that biochemical principles established for gut or terrestrial microbes also apply to marine bacteria, even though their PULs are evolutionarily distant.© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

Unraveling microbial communities associated with methylmercury production in paddy soils.

Rice consumption is now recognized as an important pathway of human exposure to the neurotoxin methylmercury (MeHg), particularly in countries where rice is a staple food. Although the discovery of a two-gene cluster hgcAB has linked Hg methylation to several phylogenetically diverse groups of anaerobic microorganisms converting inorganic mercury (Hg) to MeHg, the prevalence and diversity of Hg methylators in microbial communities of rice paddy soils remain unclear. We characterized the abundance and distribution of hgcAB genes using third-generation PacBio long-read sequencing and Illumina short-read metagenomic sequencing, in combination with quantitative PCR analyses in several mine-impacted paddy soils from southwest China. Both Illumina and PacBio sequencing analyses revealed that Hg methylating communities were dominated by iron-reducing bacteria (i.e., Geobacter) and methanogens, with a relatively low abundance of hgcA + sulfate-reducing bacteria in the soil. A positive correlation was observed between the MeHg content in soil and the relative abundance of Geobacter carrying the hgcA gene. Phylogenetic analysis also uncovered some hgcAB sequences closely related to three novel Hg methylators, Geobacter anodireducens, Desulfuromonas sp. DDH964, and Desulfovibrio sp. J2, among which G. anodireducens was validated for its ability to methylate Hg. These findings shed new light on microbial community composition and major clades likely driving Hg methylation in rice paddy soils.

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September 22, 2019

Spread of the florfenicol resistance floR gene among clinical Klebsiella pneumoniae isolates in China.

Florfenicol is a derivative of chloramphenicol that is used only for the treatment of animal diseases. A key resistance gene for florfenicol, floR, can spread among bacteria of the same and different species or genera through horizontal gene transfer. To analyze the potential transmission of resistance genes between animal and human pathogens, we investigated floR in Klebsiella pneumoniae isolates from patient samples. floR in human pathogens may originate from animal pathogens and would reflect the risk to human health of using antimicrobial agents in animals.PCR was used to identify floR-positive strains. The floR genes were cloned, and the minimum inhibitory concentrations (MICs) were determined to assess the relative resistance levels of the genes and strains. Sequencing and comparative genomics methods were used to analyze floR gene-related sequence structure as well as the molecular mechanism of resistance dissemination.Of the strains evaluated, 20.42% (67/328) were resistant to florfenicol, and 86.96% (20/23) of the floR-positive strains demonstrated high resistance to florfenicol with MICs =512 µg/mL. Conjugation experiments showed that transferrable plasmids carried the floR gene in three isolates. Sequencing analysis of a plasmid approximately 125 kb in size (pKP18-125) indicated that the floR gene was flanked by multiple copies of mobile genetic elements. Comparative genomics analysis of a 9-kb transposon-like fragment of pKP18-125 showed that an approximately 2-kb sequence encoding lysR-floR-virD2 was conserved in the majority (79.01%, 83/105) of floR sequences collected from NCBI nucleotide database. Interestingly, the most similar sequence was a 7-kb fragment of plasmid pEC012 from an Escherichia coli strain isolated from a chicken.Identified on a transferable plasmid in the human pathogen K. pneumoniae, the floR gene may be disseminated through horizontal gene transfer from animal pathogens. Studies on the molecular mechanism of resistance gene dissemination in different bacterial species of animal origin could provide useful information for preventing or controlling the spread of resistance between animal and human pathogens.

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September 22, 2019

Insights into the microbiota of Asian seabass (Lates calcarifer) with tenacibaculosis symptoms and description of sp. nov. Tenacibaculum singaporense

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the Bacteroidetes genus Tenacibaculum, most notably T. maritimum. The impact of tenacibaculosis on fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissue types of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farm fish. The microbiota of diseased farm fish was dominated by Proteobacteria (relative abundancetextpmstandard deviation, 74.5%textpm22.8%) and Bacteroidetes (18.07%textpm21.7%), the latter mainly comprised by a high abundance of Tenacibaculum species (17.6%textpm20.7%). In healthy seabass Proteobacteria had also highest relative abundance (48.04%textpm0.02%), but Firmicutes (34.2%textpm0.02%) and Fusobacteria (12.0%textpm0.03%) were the next two major constituents. Experimentally infected fish developed lesions characteristic for tenacibaculosis, but the microbiota was primarily dominated by Proteobacteria (90.4%textpm0.2%) and Firmicutes (6.2%textpm0.1%). The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in the commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One strain was isolated and is described here as sp. nov. Tenacibaculum singaporense TLL-A1T (=DSM 106434T, KCTC 62393T). The genome of T. singaporense was sequenced and compared to those of T. maritimum DSM 17995T and the newly sequenced T. mesophilum DSM 13764T.

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September 22, 2019

Genomic and metatranscriptomic analyses of Weissella koreensis reveal its metabolic and fermentative features during kimchi fermentation

The genomic and metabolic features of Weissella koreensis, one of the major lactic acid bacteria in kimchi, were investigated through genomic, metabolic, and transcriptomic analyses for the genomes of strains KCTC 3621T, KACC 15510, and WiKim0080. W. koreensis strains were intrinsically vancomycin-resistant and harbored potential hemolysin genes that were actively transcribed although no hemolysin activity was detected. KEGG and reconstructed fermentative metabolic pathways displayed that W. koreensis strains commonly employ the heterolactic pathway to produce d-lactate, ethanol, acetate, CO2, d-sorbitol, thiamine, and folate from various carbohydrates including d-glucose, d-mannose, d-lactose, l-malate, d-xylose, l-arabinose, d-ribose, N-acetyl-glucosamine, and gluconate, and strains KCTC 3621T and WiKim0080 additionally have metabolic pathways of d-galacturonate and d-glucoronate. Phenotypic analyses showed that all strains did not ferment d-galactose, probably due to the lack of d-galactose transporting system, and strains KCTC 3621T and WiKim0080 fermented d-fructose, indicating the presence of d-fructose transporting system. Fermentative features of W. koreensis were investigated through kimchi transcriptional analysis, suggesting that W. koreensis is mainly responsible for kimchi fermentation with the production of various fermentative metabolites during late fermentation period. This was the first study to investigate the genomic and metabolic features of W. koreensis, which may provide better understandings on kimchi fermentation.

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September 22, 2019

Genomic characterization of carbapenemase-producing Klebsiella pneumoniae with chromosomally encoded blaNDM-1.

We report here Klebsiella pneumoniae strains carrying chromosomal blaNDM-1 in Thailand. The genomes of these two isolates include a 160-kbp insertion containing blaNDM-1, which is almost identical to that in the IncHI1B-like plasmid. Further analysis indicated that IS5-mediated intermolecular transposition and Tn3 transposase-mediated homologous recombination resulted in the integration of blaNDM-1 into the chromosome from an IncHI1B-like plasmid. The spread of this type of carbapenem-resistant Enterobacteriaceae may threaten public health and warrants further monitoring. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Description of Schaedlerella arabinophila gen. nov., sp. nov., a D-arabinose utilizing bacterium isolated from feces of C57BL/6J mice and a close relative of Clostridium sp. ASF 502

The use of gnotobiotics has gained large interest in recent years due to technological advances that have revealed the importance of host-associated microbiomes for host physiology and health. One of the oldest and most important gnotobiotics mouse model, the Altered Schaedler Flora (ASF) has been used for several decades. ASF comprises eight different bacterial species, which have been characterized to different extent, but only few are available through public strain collections. Here, the isolation of a close relative to one of the less studied ASF strains, Clostridium sp. ASF 502, is reported. Isolate TLL-A1, which shares 99.6% 16S rRNA gene sequence identity with Clostridium sp. ASF 502, was obtained from feces of C57BL/6J mice where is was detectable at a relative abundance of less than one percent. D-arabinose was used as sole carbon source in the anaerobic cultivation medium. Growth experiments with TLL-A1 on different carbon sources and analysis of its ~6.5 gigabase genome indicate that TLL-A1 harbors a large gene repertoire to utilize different carbohydrates for growth. Comparative genome analyses of TLL-A1 and Clostridium sp. ASF 502 reveal differences in genome content between the two strains, in particular with regards to carbohydrate activating enzymes. Based on physiology and genomic analysis it is proposed to name TLL-A1 to gen. nov. sp. nov Schaedlerella arabinophila TLL-A1 (DSMZ 106076T; KCTC 15657T). The closely related Clostridium sp. ASF 502 is proposed to be renamed to Schaedlerella arabinophila to reflect its taxonomic standing and to keep textquoterightASF 502textquoteright as strain designation.

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September 22, 2019

CompStor Novos: a low cost yet fast assembly-based variant calling for personal genomes

Application of assembly methods for personal genome analysis from next generation sequencing data has been limited by the requirement for an expensive supercomputer hardware or long computation times when using ordinary resources. We describe CompStor Novos, achieving supercomputer-class performance in de novo assembly computation time on standard server hardware, based on a tiered-memory algorithm. Run on commercial off-the-shelf servers, Novos assembly is more precise and 10-20 times faster than that of existing assembly algorithms. Furthermore, we integrated Novos into a variant calling pipeline and demonstrate that both compute times and precision of calling point variants and indels compare well with standard alignment-based pipelines. Additionally, assembly eliminates bias in the estimation of allele frequency for indels and naturally enables discovery of breakpoints for structural variants with base pair resolution. Thus, Novos bridges the gap between alignment-based and assembly-based genome analyses. Extension and adaption of its underlying algorithm will help quickly and fully harvest information in sequencing reads for personal genome reconstruction.

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September 22, 2019

Mutators as drivers of adaptation in Streptococcus and a risk factor for host jumps and vaccine escape

Heritable hypermutable strains deficient in DNA repair genes (mutators) facilitate microbial adaptation as they may rapidly generate beneficial mutations. Mutators deficient in mismatch (MMR) and oxidised guanine (OG) repair are abundant in clinical samples and show increased adaptive potential in experimental infection models but their role in pathoadaptation is poorly understood. Here we investigate the role of mutators in epidemiology and evolution of the broad host pathogen, Streptococcus iniae, employing 80 strains isolated globally over 40 years. We determine phylogenetic relationship among S. iniae using 10,267 non-recombinant core genome single nucleotide polymorphisms (SNPs), estimate their mutation rate by fluctuation analysis, and detect variation in major MMR (mutS, mutL, dnaN, recD2, rnhC) and OG (mutY, mutM, mutX) genes. S. iniae mutation rate phenotype and genotype are strongly associated with phylogenetic diversification and variation in major streptococcal virulence determinants (capsular polysaccharide, hemolysin, cell chain length, resistance to oxidation, and biofilm formation). Furthermore, profound changes in virulence determinants observed in mammalian isolates (atypical host) and vaccine-escape isolates found in bone (atypical tissue) of vaccinated barramundi are linked to multiple MMR and OG variants and unique mutation rates. This implies that adaptation to new host taxa, new host tissue, and to immunity of a vaccinated host is promoted by mutator strains. Our findings support the importance of mutation rate dynamics in evolution of pathogenic bacteria, in particular adaptation to a drastically different immunological setting that occurs during host jump and vaccine escape events.Importance Host immune response is a powerful selective pressure that drives diversification of pathogenic microorganisms and, ultimately, evolution of new strains. Major adaptive events in pathogen evolution, such as transmission to a new host species or infection of vaccinated hosts, require adaptation to a drastically different immune landscape. Such adaptation may be favoured by hypermutable strains (or mutators) that are defective in normal DNA repair and consequently capable of generating multiple potentially beneficial and compensatory mutations. This permits rapid adjustment of virulence and antigenicity in a new immunological setting. Here we show that mutators, through mutations in DNA repair genes and corresponding shifts in mutation rate, are associated with major diversification events and virulence evolution in the broad host-range pathogen Streptococcus iniae. We show that mutators underpin infection of vaccinated hosts, transmission to new host species and the evolution of new strains.

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September 21, 2019

Chromulinavorax destructans, a pathogenic TM6 bacterium with an unusual replication strategy targeting protist mitochondrion

Most of the diversity of microbial life is not available in culture, and as such we lack even a fundamental understanding of the biological diversity of several branches on the tree of life. One branch that is highly underrepresented is the candidate phylum TM6, also known as the Dependentiae. Their biology is known only from reduced genomes recovered from metagenomes around the world and two isolates infecting amoebae, all suggest that they live highly host-associated lifestyles as parasites or symbionts. Chromulinavorax destructans is an isolate from the TM6/Dependentiae that infects and lyses the abundant heterotrophic flagellate, Spumella elongata. Chromulinavorax destructans is characterized by a high degree of reduction and specialization for infection, so much so it was discovered in a screen for giant viruses. Its 1.2 Mb genome shows no metabolic potential and C. destructans instead relies on extensive transporter system to import nutrients, and even energy in the form of ATP from the host. Accordingly, it replicates in a viral-like fashion, while extensively reorganizing and expanding the host mitochondrion. 44% of proteins contain signal sequences for secretion, which includes many proteins of unknown function as well as 98 copies of ankyrin-repeat domain proteins, known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus.

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