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Asset Tag: HGAP

September 22, 2019

Complete genome sequence of blaIMP-6-positive Metakosakonia sp. MRY16-398 isolate from the ascites of a diverticulitis patient.

A novel species of carbapenemase-producing Enterobacteriaceae (CPE) was isolated from a patient diagnosed with sigmoid colon diverticulitis. At first, laboratory testing suggested it was Klebsiella oxytoca or Pantoea sp.; however, a complete genome sequence of the isolate, MRY16-398, revealed that it could be novel species, most similar to [Kluyvera] intestini, of which taxonomic nomenclature is still under discussion. Orthologous conserved gene analysis among 42 related bacterial strains indicated that MRY16-398 was classified as the newly proposed genus Metakosakonia. Further, MRY16-398 was found to harbor the blaIMP-6 gene-positive class 1 integron (In722) in plasmid pMRY16-398_2 (IncN replicon, 47.4 kb in size). This finding implies that rare and opportunistic bacteria could be potential infectious agents. In conclusion, our results highlight the need for continuous monitoring for CPE even in nonpathogenic bacteria in the nosocomial environment.

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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

Complete Genome Sequence of Massilia oculi sp. nov. CCUG 43427T (=DSM 26321T), the Type Strain of M. oculi, and Comparison with Genome Sequences of Other Massilia Strains.

Massilia oculi sp. nov. of type strain CCUG 43427T is a Gram-negative, rod-shaped, nonspore-forming bacterium, which was recently isolated from the eye of a patient suffering from endophthalmitis and was described as novel species in Massilia genus. In this study, we present the complete genome sequence of this strain by using Pacbio SMRT cell platform and compare this sequence with the genomes of 30 Massilia representative strains. Also, a comprehensive search was conducted for genes and proteins involved in antibiotic resistance and pathogenicity. The genome of CCUG 43427T is 5,844,653 bp with 65.55% GC content. This genome contains four prophages and four genomic islands (GIs). The cobalt/zinc/cadmium transporter locus CzcABCD is included in these GIs. This GI was predicted to play important role in bacterial heavy-metal tolerance. The in silico genome analysis also revealed that this strain contains a lot of antibiotic resistance and pathogenicity related genes. This result suggested that this strain may has evolved a wide arsenal of weapons for pathogenicity and survival. Genome comparison among CCUG 43427T and other 30 Massilia strains revealed that more than 400 genes are unique in CCUG 43427T. Among these, one gene cluster, which was annotated to be important for LOS biosynthesis, catalytic mechanism and the substrate specificity of the enzyme, was predicted to be horizontally transferred by using phylogenies and biased GC content.

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September 22, 2019

Identification of DNA base modifications by means of Pacific Biosciences RS Sequencing technology.

Whole phage genomes can be sequenced readily using one or a combination of next generation sequencing (NGS) technologies. One of the most recently developed NGS platforms, the so-called Single-Molecule Real-Time (SMRT) sequencing approach provided by the PacBio RS platform, is particularly useful in providing complete (i.e., un-gapped) genome sequences, but differs from other technologies in that the platform also allows for downstream analysis to identify nucleotides that have been modified by DNA methylation. Here, we describe the methodological approach for the detection of genomic methylation motifs by means of SMRT sequencing.

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September 22, 2019

Construction of stable fluorescent laboratory control strains for several food safety relevant Enterobacteriaceae.

Using naturally-occurring bacterial strains as positive controls in testing protocols is typically feared due to the risk of cross-contaminating samples. We have developed a collection of strains which express Green Fluorescent Protein (GFP) at high-level, permitting rapid screening of the following species on selective or non-selective plates: Escherichia coli O157:H7, Shigella sonnei, S. flexneri, Salmonella enterica subsp. Enterica serovar Gaminera, S. Mbandaka, S. Tennesse, S. Minnesota, S. Senftenberg and S. Typhimurium. These new strains fluoresce when irradiated with UV light and maintain this phenotype in absence of antibiotic selection. Recombinants were phenotypically equivalent to the parent strain, except for S. Tennessee Sal66 that appeared Lac- on Xylose Lysine Deoxycholate (XLD) agar plates and Lac+ on Mac Conkey and Hektoen Enteric agar plates. Analysis of closed whole genome sequences revealed that Sal66 had lost one lactose operon; slower rates of lactose metabolism may affect lactose fermentation on XLD agar. These fluorescent enteric control strains were challenging to develop and should provide an easy and effective means of identifying cross-contamination. Published by Elsevier Ltd.

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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

Comparative genomic analysis revealed rapid differentiation in the pathogenicity-related gene repertoires between Pyricularia oryzae and Pyricularia penniseti isolated from a Pennisetum grass.

A number of Pyricularia species are known to infect different grass species. In the case of Pyricularia oryzae (syn. Magnaporthe oryzae), distinct populations are known to be adapted to a wide variety of grass hosts, including rice, wheat and many other grasses. The genome sizes of Pyricularia species are typical for filamentous ascomycete fungi [~?40 Mbp for P. oryzae, and ~?45 Mbp for P. grisea]. Genome plasticity, mediated in part by deletions promoted by recombination between repetitive elements [Genome Res 26:1091-1100, 2016, Nat Rev Microbiol 10:417-430,2012] and transposable elements [Annu Rev Phytopathol 55:483-503,2017] contributes to host adaptation. Therefore, comparisons of genome structure of individual species will provide insight into the evolution of host specificity. However, except for the P. oryzae subgroup, little is known about the gene content or genome organization of other Pyricularia species, such as those infecting Pennisetum grasses.Here, we report the genome sequence of P. penniseti strain P1609 isolated from a Pennisetum grass (JUJUNCAO) using PacBio SMRT sequencing technology. Phylogenomic analysis of 28 Magnaporthales species and 5 non-Magnaporthales species indicated that P1609 belongs to a Pyricularia subclade, which is genetically distant from P. oryzae. Comparative genomic analysis revealed that the pathogenicity-related gene repertoires had diverged between P1609 and the P. oryzae strain 70-15, including the known avirulence genes, other putative secreted proteins, as well as some other predicted Pathogen-Host Interaction (PHI) genes. Genomic sequence comparison also identified many genomic rearrangements relative to P. oryzae.Our results suggested that the genomic sequence of the P. penniseti P1609 could be a useful resource for the genetic study of the Pennisetum-infecting Pyricularia species and provide new insight into evolution of pathogen genomes during host adaptation.

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September 22, 2019

Meiotic drive of female-inherited supernumerary chromosomes in a pathogenic fungus.

Meiosis is a key cellular process of sexual reproduction that includes pairing of homologous sequences. In many species however, meiosis can also involve the segregation of supernumerary chromosomes, which can lack a homolog. How these unpaired chromosomes undergo meiosis is largely unknown. In this study we investigated chromosome segregation during meiosis in the haploid fungus Zymoseptoria tritici that possesses a large complement of supernumerary chromosomes. We used isogenic whole chromosome deletion strains to compare meiotic transmission of chromosomes when paired and unpaired. Unpaired chromosomes inherited from the male parent as well as paired supernumerary chromosomes in general showed Mendelian inheritance. In contrast, unpaired chromosomes inherited from the female parent showed non-Mendelian inheritance but were amplified and transmitted to all meiotic products. We concluded that the supernumerary chromosomes of Z. tritici show a meiotic drive and propose an additional feedback mechanism during meiosis, which initiates amplification of unpaired female-inherited chromosomes.© 2018, Habig et al.

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September 22, 2019

Complete genome sequence of Leuconostoc citreum EFEL2700, a host strain for transformation of pCB vectors.

Leuconostoc citreum is an important lactic acid bacterium used as a starter culture for producing kimchi, the traditional Korean fermented vegetables. An efficient host strain for plasmid transformation, L. citreum EFEL2700, was isolated from kimchi, and it has been frequently used for genetic engineering of L. citreum. In this study, we report the whole genome sequence of the strain and its genetic characteristics. Genome assembly yielded 5 contigs (1 chromosome and 4 plasmids), and the complete genome contained 1,923,830 base pairs (bp) with a G?+?C content of 39.0%. Average nucleotide identity analysis showed high homology (= 99%) to the reference strain L. citreum KM 20. The smallest plasmid (4.3 kbp) was used as an Escherichia coli shuttle vector (pCB) for heterologous gene expression, and L. citreum EFEL2700 showed the highest transformation efficiency, 6.7?×?104 CFU µg-1 DNA. Genetic analysis of the genome enabled the construction of primary metabolic pathway showing a typical hetero-type lactic acid fermentation. Notably, no core genes for primary metabolism were observed in plasmid 4 and it could be eliminated to create an efficient host for gene transformation. This report will facilitate the understanding and application of L. citreum EFEL2700 as a food-grade microbial cell factory.Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019

Novel linezolid resistance plasmids in Enterococcus from food animals in the USA.

To sequence the genomes and determine the genetic mechanisms for linezolid resistance identified in three strains of Enterococcus isolated from cattle and swine caecal contents as part of the US National Antimicrobial Resistance Monitoring System (NARMS) surveillance programme.Broth microdilution was used for in vitro antimicrobial susceptibility testing to assess linezolid resistance. Resistance mechanisms and plasmid types were identified from data generated by WGS on Illumina® and PacBio® platforms. Conjugation experiments were performed to determine whether identified mechanisms were transmissible.Linezolid resistance plasmids containing optrA were identified in two Enterococcus faecalis isolates and one Enterococcus faecium. The E. faecium isolate also carried the linezolid resistance gene cfr on the same plasmid as optrA. The linezolid resistance plasmids had various combinations of additional resistance genes conferring resistance to phenicols (fexA), aminoglycosides [spc and aph(3′)-III] and macrolides [erm(A) and erm(B)]. One of the plasmids was confirmed to be transmissible by conjugation, resulting in linezolid resistance in the transconjugant.To the best of our knowledge, this is the first identification of linezolid resistance in the USA in bacteria isolated from food animals. The oxazolidinone class of antibiotics is not used in food animals in the USA, but the genes responsible for resistance were identified on plasmids with other resistance markers, indicating that there may be co-selection for these plasmids due to the use of different antimicrobials. The transmissibility of one of the plasmids demonstrated the potential for linezolid resistance to spread horizontally. Additional surveillance is necessary to determine whether similar plasmids are present in human strains of Enterococcus.

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September 22, 2019

Implications of stx loss for clinical diagnostics of Shiga toxin-producing Escherichia coli.

The dynamics related to the loss of stx genes from Shiga toxin-producing Escherichia coli remain unclear. Current diagnostic procedures have shortcomings in the detection and identification of STEC. This is partly owing to the fact that stx genes may be lost during an infection or in the laboratory. The aim of the present study was to provide new insight into in vivo and in vitro stx loss in order to improve diagnostic procedures. Results from the study support the theory that loss of stx is a strain-related phenomenon and not induced by patient factors. It was observed that one strain could lose stx both in vivo and in vitro. Whole genome comparison of stx-positive and stx-negative isolates from the same patient revealed that different genomic rearrangements, such as complete or partial loss of the parent prophage, may be factors in the loss of stx. Of diagnostic interest, it was shown that patients can be co-infected with different E. coli pathotypes. Therefore, identification of eae-positive, but stx-negative isolates should not be interpreted as “Shiga toxin-lost” E. coli without further testing. Growth and recovery of STEC were supported by different selective agar media for different strains, arguing for inclusion of several media in STEC diagnostics.

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September 22, 2019

Genome-scale analysis of Acetobacterium bakii reveals the cold adaptation of psychrotolerant acetogens by post-transcriptional regulation.

Acetogens synthesize acetyl-CoA via CO2 or CO fixation, producing organic compounds. Despite their ecological and industrial importance, their transcriptional and post-transcriptional regulation has not been systematically studied. With completion of the genome sequence of Acetobacterium bakii (4.28-Mb), we measured changes in the transcriptome of this psychrotolerant acetogen in response to temperature variations under autotrophic and heterotrophic growth conditions. Unexpectedly, acetogenesis genes were highly up-regulated at low temperatures under heterotrophic, as well as autotrophic, growth conditions. To mechanistically understand the transcriptional regulation of acetogenesis genes via changes in RNA secondary structures of 5′-untranslated regions (5′-UTR), the primary transcriptome was experimentally determined, and 1379 transcription start sites (TSS) and 1100 5′-UTR were found. Interestingly, acetogenesis genes contained longer 5′-UTR with lower RNA-folding free energy than other genes, revealing that the 5′-UTRs control the RNA abundance of the acetogenesis genes under low temperature conditions. Our findings suggest that post-transcriptional regulation via RNA conformational changes of 5′-UTRs is necessary for cold-adaptive acetogenesis.© 2018 Shin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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September 22, 2019

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri.

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.

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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

Discovery of the actinoplanic acid pathway in Streptomyces rapamycinicus reveals a genetically conserved synergism with rapamycin.

Actinobacteria possess a great wealth of pathways for production of bioactive compounds. Following advances in genome mining, dozens of natural product (NP) gene clusters are routinely found in each actinobacterial genome; however, the modus operandi of this large arsenal is poorly understood. During investigations of the secondary metabolome of Streptomyces rapamycinicus, the producer of rapamycin, we observed accumulation of two compounds never before reported from this organism. Structural elucidation revealed actinoplanic acid A and its demethyl analogue. Actinoplanic acids (APLs) are potent inhibitors of Ras farnesyltransferase and therefore represent bioactive compounds of medicinal interest. Supported with the unique structure of these polyketides and using genome mining, we identified a gene cluster responsible for their biosynthesis in S. rapamycinicus Based on experimental evidence and genetic organization of the cluster, we propose a stepwise biosynthesis of APL, the first bacterial example of a pathway incorporating the rare tricarballylic moiety into an NP. Although phylogenetically distant, the pathway shares some of the biosynthetic principles with the mycotoxins fumonisins. Namely, the core polyketide is acylated with the tricarballylate by an atypical nonribosomal peptide synthetase-catalyzed ester formation. Finally, motivated by the conserved colocalization of the rapamycin and APL pathway clusters in S. rapamycinicus and all other rapamycin-producing actinobacteria, we confirmed a strong synergism of these compounds in antifungal assays. Mining for such evolutionarily conserved coharboring of pathways would likely reveal further examples of NP sets, attacking multiple targets on the same foe. These could then serve as a guide for development of new combination therapies.© 2018 Mrak et al.

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