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Asset Tag: Microbiology

April 21, 2020

The mating system of the Eucalyptus canker pathogen Chrysoporthe austroafricana and closely related species.

Fungi in the genus Chrysoporthe are economically important canker pathogens of commercially grown Eucalyptus species and native Myrtales. Before the current study, homothallism was widely accepted as the mating system of these species, but this hypothesis could not be fully tested. Using whole genome sequences, we characterized the MAT locus of two C. austroafricana isolates and its sibling species, C. cubensis and C. deuterocubensis. A unique MAT1-2 idiomorph containing a truncated MAT1-1-1 gene, and a MAT1-1-2 gene, was identified in one isolate of C. austroafricana and a MAT1-1 idiomorph was found in the other. The presence of a single idiomorph in each isolate suggests that this fungus is heterothallic. Screening for MAT genes in 65?C. austroafricana isolates revealed a bias towards MAT1-2 idiomorphs suggesting a recent introduction in Eucalyptus species. Chrysoporthe cubensis and C. deuterocubensis are apparently homothallic since all the expected MAT genes were identified in their genome sequences. These findings were corroborated by the expression profiles of pheromone genes and their receptors, which conformed to the expected patterns observed in heterothallic and homothallic isolates. Long terminal repeat sequences (LTRs) and specifically retrotransposons were identified in the MAT locus of C. deuterocubensis and C. cubensis, indicating that the evolution of mating systems in Chrysoporthe species could be mediated by these elements.Copyright © 2018 Elsevier Inc. All rights reserved.

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April 21, 2020

Antimicrobial, plant growth-promoting and genomic properties of the peanut endophyte Bacillus velezensis LDO2.

Peanut suffer from a number of fungal and bacterial pathogens, while plant endophytes were considered excellent candidates as biocontrol agents. In this study, the peanut endophytic bacterium LDO2 was evaluated for the potential of peanut pathogens inhibition and growth-promotion, and the genetic mechanisms were explored by genome mining. Strain LDO2 significantly inhibited the growth of peanut pathogenic fungi and pathogenic bacteria, and specifically, it showed pronounced inhibition on mycelia growth of Aspergillus flavus mycelia and caused mycelial deformity. Gene clusters responsible for antifungal metabolites (fengycin, surfactin, bacilysin) and antibacterial metabolites (butirosin, bacillaene, difficidin, macrolactin, surfactin, bacilysin) were identified. Strain LDO2 also exhibited several growth-promoting related features including phosphate solubilization, siderophore production and growth promotion of peanut root. Genes associated with plant growth promotion were also identified and analyzed, as well as genes related to secreted proteins. These findings suggested that this peanut endophyte could be a potential biocontrol agent in peanut production and a source of antimicrobial compounds for further exploitation. Copyright © 2018 Elsevier GmbH. All rights reserved.

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April 21, 2020

Distribution and characterization of N-acylhomoserine lactone (AHL)-degrading activity and AHL lactonase gene (qsdS) in Sphingopyxis.

N-Acylhomoserine lactone (AHL)-degrading enzyme is identified from the various environments and applied for quorum-sensing inhibition. In this study, we isolated two AHL-degrading strains, Sphingopyxis sp. EG6 and FD7, from the industrial cooling water samples. When the eight Sphingopyxis type strains were checked for the AHL-degrading activity, two strains, Sphingopyxis alaskensis DSM 13593 and Sphingopyxis bauzanensis DSM 22271, showed high AHL-degrading activity. The complete genome sequences of EG6 and FD7 revealed the presence of gene homolog of qsdS, which encodes AHL-lactonase in Sphingomonas ursincola. The qsdS gene is seated between putative gene homologs involved in 3-isopropylmalate dehydratase large (leuC2) and small (leuD) subunits in the genome of EG6, FD7, DSM 13593, and DSM 22271, but completely disappeared between leuC2 and leuD in the genome sequences of Sphingopyxis type strains without AHL-degrading activity. Purified His-tagged QsdS showed high AHL-degrading activity and catalyzed AHL ring opening by hydrolyzing lactones. In addition, heterologous expression of qsdS in Pseudomonas aeruginosa resulted in reduction of biofilm formation. These results suggested that the AHL-degrading activity in Sphingopyxis is useful as an effective agent for biofilm inhibition.Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

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April 21, 2020

In-depth analysis of the genome of Trypanosoma evansi, an etiologic agent of surra.

Trypanosoma evansi is the causative agent of the animal trypanosomiasis surra, a disease with serious economic burden worldwide. The availability of the genome of its closely related parasite Trypanosoma brucei allows us to compare their genetic and evolutionarily shared and distinct biological features. The complete genomic sequence of the T. evansi YNB strain was obtained using a combination of genomic and transcriptomic sequencing, de novo assembly, and bioinformatic analysis. The genome size of the T. evansi YNB strain was 35.2 Mb, showing 96.59% similarity in sequence and 88.97% in scaffold alignment with T. brucei. A total of 8,617 protein-coding genes, accounting for 31% of the genome, were predicted. Approximately 1,641 alternative splicing events of 820 genes were identified, with a majority mediated by intron retention, which represented a major difference in post-transcriptional regulation between T. evansi and T. brucei. Disparities in gene copy number of the variant surface glycoprotein, expression site-associated genes, microRNAs, and RNA-binding protein were clearly observed between the two parasites. The results revealed the genomic determinants of T. evansi, which encoded specific biological characteristics that distinguished them from other related trypanosome species.

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April 21, 2020

Genome-wide Transcript Structure Resolution Reveals Abundant Alternate Isoform Usage from Murine Gammaherpesvirus 68.

The gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, MuHV-4, ?HV68), are etiologic agents of a wide range of lymphomas and non-hematological malignancies. These viruses possess large and highly dense dsDNA genomes that feature >80 bidirectionally positioned open reading frames (ORFs). The abundance of overlapping transcripts and extensive splicing throughout these genomes have until now prohibited high throughput-based resolution of transcript structures. Here, we integrate the capabilities of long-read sequencing with the accuracy of short-read platforms to globally resolve MHV68 transcript structures using the transcript resolution through integration of multi-platform data (TRIMD) pipeline. This approach reveals highly complex features, including: (1) pervasive overlapping transcript structures; (2) transcripts containing intra-gene or trans-gene splices that yield chimeric ORFs; (3) antisense and intergenic transcripts containing ORFs; and (4) noncoding transcripts. This work sheds light on the underappreciated complexity of gammaherpesvirus transcription and provides an extensively revised annotation of the MHV68 transcriptome. Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Evolution and transmission of a conjugative plasmid encoding both ciprofloxacin and ceftriaxone resistance in Salmonella.

Ceftriaxone and ciprofloxacin are the drugs of choice in treatment of invasive Salmonella infections. This study discovered a novel type of plasmid, pSa44-CIP-CRO, which was recovered from a S. London strain isolated from meat product and comprised genetic determinants that encoded resistance to both ciprofloxacin and ceftriaxone. This plasmid could be resolved into two daughter plasmids and co-exist with such daughter plasmids in a dynamic form in Salmonella; yet it was only present as a single plasmid in Escherichia coli. One daughter plasmid, pSa44-CRO, was found to carry the blaCTX-M-130 gene, which encodes resistance to ceftriaxone, whereas the other plasmid, pSa44-CIP, carried multiple PMQR genes such as qnrB6-aac(6′)-Ib-cr, which mediated resistance to ciprofloxacin. These two daughter plasmids could be integrated into one single plasmid through ISPa40 mediated homologous recombination. Mouse infection and treatment experiments showed that carriage of plasmid, pSa44-CIP-CRO by S. typhimurium led to the impairment of treatment by ciprofloxacin or cefitiofur, a veterinary drug with similar properties as ceftriaxone. In conclusion, dissemination of such conjugative plasmids impairs current choices of treatment for life-threatening Salmonella infection and hence constitutes a serious public health threat.

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April 21, 2020

Bioinformatic analysis of the complete genome sequence of Pectobacterium carotovorum subsp. brasiliense BZA12 and candidate effector screening

AbstractPectobacterium carotovorum subsp. brasiliense (Pcb) is a gram-negative, plant pathogenic bacterium of the soft rot Enterobacteriaceae (SRE) family. We present the complete genome sequence of Pcb strain BZA12, which reveals that Pcb strain BZA12 carries a single 4,924,809 bp chromosome with 51.97% GC content and comprises 4508 predicted protein-coding genes.Geneannotationofthese genes utilizedGO, KEGG,and COG databases.Incomparison withthree closely related soft-rot pathogens, strain BZA12 has 3797 gene families, among which 3107 gene families are identified as orthologous with those of both P. carotovorum subsp. carotovorum PCC21 and P. carotovorum subsp. odoriferum BCS7, as well as 36 putative Unique Gene Families. We selected five putative effectors from the BZA12 genome and transiently expressed them in Nicotiana benthamiana. Candidate effector A12GL002483 was localized in the cell nucleus and induced cell death. This study provides a foundation for a better understanding of the genomic structure and function of Pcb, particularly in the discovery of potential pathogenic factors and for the development of more effective strategies against this pathogen.

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April 21, 2020

Physiological properties and genetic analysis related to exopolysaccharide (EPS) production in the fresh-water unicellular cyanobacterium Aphanothece sacrum (Suizenji Nori).

The clonal strains, phycoerythrin(PE)-rich- and PE-poor strains, of the unicellular, fresh water cyanobacterium Aphanothece sacrum (Suringar) Okada (Suizenji Nori, in Japanese) were isolated from traditional open-air aquafarms in Japan. A. sacrum appeared to be oligotrophic on the basis of its growth characteristics. The optimum temperature for growth was around 20°C. Maximum growth and biomass increase at 20°C was obtained under light intensities between 40 to 80 µmol m-2 s-1 (fluorescent lamps, 12 h light/12 h dark cycles) and between 40 to 120 µmol m-2 s-1 for PE-rich and PE-poor strains, respectively, of A. sacrum . Purified exopolysaccharide (EPS) of A. sacrum has a molecular weight of ca. 104 kDa with five major monosaccharides (glucose, xylose, rhamnose, galactose and mannose; =85 mol%). We also deciphered the whole genome sequence of the two strains of A. sacrum. The putative genes involved in the polymerization, chain length control, and export of EPS would contribute to understand the biosynthetic process of their extremely high molecular weight EPS. The putative genes encoding Wzx-Wzy-Wzz- and Wza-Wzb-Wzc were conserved in the A. sacrum strains FPU1 and FPU3. This result suggests that the Wzy-dependent pathway participates in the EPS production of A. sacrum.

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April 21, 2020

Whole genome sequencing used in an industrial context reveals a Salmonella laboratory cross-contamination.

In 2013, during a routine laboratory analysis performed on food samples, one finished product from a European factory was tested positive for Salmonella Hadar. At the same period, one environmental isolate in the same laboratory was serotyped Salmonella Hadar. Prior to this event, the laboratory performed a proficiency testing involving a sample spiked with NCTC 9877 Salmonella Hadar. The concomitance of Salmonella Hadar detection led to the suspicion of a laboratory cross-contamination between the Salmonella Hadar isolate used in the laboratory proficiency testing and the Salmonella Hadar isolate found on the finished product by the same laboratory. Since the classical phenotypic serotyping method is able to attribute a serotype to Salmonella isolates with a common antigenic formula, but cannot differentiate strains of the same serotype within the subspecies, whole genome sequencing was used to test the laboratory cross-contamination hypothesis. Additionally, 12 Salmonella Hadar from public databases, available until the time of the event, were included in the whole genome sequencing analysis to better understand the genomic diversity of this serotype in Europe. The outcome of the analysis showed a maximum of ten single nucleotide polymorphisms (SNPs) between the isolates coming from the laboratory and the finished product, and thus confirmed the laboratory cross-contamination. These results combined with all additional investigations done at the factory, allowed to release finished product batches produced and thus circumvented unnecessary food waste and economic losses for the factory. Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.

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April 21, 2020

Investigating the bacterial microbiota of traditional fermented dairy products using propidium monoazide with single-molecule real-time sequencing.

Traditional fermented dairy foods have been the major components of the Mongolian diet for millennia. In this study, we used propidium monoazide (PMA; binds to DNA of nonviable cells so that only viable cells are enumerated) and single-molecule real-time sequencing (SMRT) technology to investigate the total and viable bacterial compositions of 19 traditional fermented dairy foods, including koumiss from Inner Mongolia (KIM), koumiss from Mongolia (KM), and fermented cow milk from Mongolia (CM); sample groups treated with PMA were designated PKIM, PKM, and PCM. Full-length 16S rRNA sequencing identified 195 bacterial species in 121 genera and 13 phyla in PMA-treated and untreated samples. The PMA-treated and untreated samples differed significantly in their bacterial community composition and a-diversity values. The predominant species in KM, KIM, and CM were Lactobacillus helveticus, Streptococcus parauberis, and Lactobacillus delbrueckii, whereas the predominant species in PKM, PKIM, and PCM were Enterobacter xiangfangensis, Lactobacillus helveticus, and E. xiangfangensis, respectively. Weighted and unweighted principal coordinate analyses showed a clear clustering pattern with good separation and only minor overlapping. In addition, a pure culture method was performed to obtain lactic acid bacteria resources in dairy samples according to the results of SMRT sequencing. A total of 102 LAB strains were identified and Lb. helveticus (68.63%) was the most abundant, in agreement with SMRT sequencing results. Our results revealed that the bacterial communities of traditional dairy foods are complex and vary by type of fermented dairy product. The PMA treatment induced significant changes in bacterial community structure.Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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April 21, 2020

Potential use of the Pteris vittata arsenic hyperaccumulation-regulation network for phytoremediation.

Arsenic accumulation in soil is a global problem typically addressed using phytoremediation methods. Pteris vittata, a model arsenic hyperaccumulator, has great potential as a genetically engineered plant for phytoremediation. However, the lack of omic information on this species has severely limited the identification and application of its arsenic hyperaccumulation and regulation components. In this study, we used an optimized single-molecular real-time (SMRT) strategy to create a de novo full-length transcriptomic-tonoplast proteomic database for this unsequenced fern and to determine the genetic components underlying its arsenic hyperaccumulation-regulation mechanisms. We established a comprehensive network consisting of six major transporter families, two novel resistance pathways, and a regulatory system by examining alternative splicing (AS) and long non-coding RNA (lncRNA) in different tissues following As(III) and As(V) treatment. The database and network established in this study will deepen our understanding of the unique hyperaccumulation and regulation mechanisms of P. vittata, ultimately providing a valuable resource for futher research on phytoremediation of arsenic-contaminated soil. Copyright © 2019 Elsevier B.V. All rights reserved.

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April 21, 2020

Mediterraneibacter butyricigenes sp. nov., a butyrate-producing bacterium isolated from human faeces.

A Gram-stain-positive, obligately anaerobic, non-motile, nonspore-forming, and rod-shaped bacterial strain, designated KGMB01110T, was isolated from a faecal sample of a healthy male in South Korea. Phylogenetic analysis based on 16S rRNA gene showed that strain KGMB01110T belonged to Clostridium cluster XIVa and was most closely related to Mediterraneibacter glycyrrhizinilyticus KCTC 5760T (95.9% 16S rRNA gene sequence similarity). The DNA G + C content of strain KGMB01110T based on its whole genome sequence was 44.1 mol%. The major cellular fatty acids (> 10%) of the isolate were C14:0 and C16:0. The strain KGMB01110T was positive for arginine dihydrolase, ß-galactosidase-6-phosphatase, and alkaline phosphatase. The strain KGMB01110T also produced acid from D-glucose and D-rhamnose, and hydrolyzed gelatin and aesculin. Furthermore, HPLC analysis and UV-tests of culture supernatant revealed that the strain KGMB01110T produced butyrate as the major end product of glucose fermentation. Based on the phylogenetic and phenotypic characteristics, strain KGMB01110T represent a novel species of the genus Mediterraneibacter in the family Lachnospiraceae. The type strain is KGMB01110T (= KCTC 15684T = CCUG 72830T).

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April 21, 2020

Novel trimethoprim resistance gene dfrA34 identified in Salmonella Heidelberg in the USA.

Trimethoprim/sulfamethoxazole is a synthetic antibiotic combination recommended for the treatment of complicated non-typhoidal Salmonella infections in humans. Resistance to trimethoprim/sulfamethoxazole is mediated by the acquisition of mobile genes, requiring both a dfr gene (trimethoprim resistance) and a sul gene (sulfamethoxazole resistance) for a clinical resistance phenotype (MIC =4/76?mg/L). In 2017, the CDC investigated a multistate outbreak caused by a Salmonella enterica serotype Heidelberg strain with trimethoprim/sulfamethoxazole resistance, in which sul genes but no known dfr genes were detected.To characterize and describe the molecular mechanism of trimethoprim resistance in a Salmonella Heidelberg outbreak isolate.Illumina sequencing data for one outbreak isolate revealed a 588?bp ORF encoding a putative dfr gene. This gene was cloned into Escherichia coli and resistance to trimethoprim was measured by broth dilution and Etest. Phylogenetic analysis of previously reported dfrA genes was performed using MEGA. Long-read sequencing was conducted to determine the context of the novel dfr gene.The novel dfr gene, named dfrA34, conferred trimethoprim resistance (MIC =32?mg/L) when cloned into E. coli. Based on predicted amino acid sequences, dfrA34 shares less than 50% identity with other known dfrA genes. The dfrA34 gene is located in a class 1 integron in a multiresistance region of an IncC plasmid, adjacent to a sul gene, thus conferring clinical trimethoprim/sulfamethoxazole resistance. Additionally, dfrA34 is associated with ISCR1, enabling easy transmission between other plasmids and bacterial strains.

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April 21, 2020

Intestinibaculum porci gen. nov., sp. nov., a new member of the family Erysipelotrichaceae isolated from the small intestine of a swine.

A strictly anaerobic, Gram-stain-positive, catalase-negative, non-motile, rod-shaped bacterium, designated SG0102T, was isolated from the small intestine of a swine. Optimal growth occurred at 37°C and pH 7.0. Furthermore, growth was observed in the presence of up to 3% (w/v) NaCl but not at salinity levels higher than 4%. The comparative analysis of 16S rRNA gene sequences showed that strain SG0102T was most closely related to Kandleria vitulina DSM 20405T (93.3%), followed by Catenibacterium mitsuokai KCTC 5053T (91.1%), Sharpea azabuensis KCTC 15217T (91.0%), and Eggerthia catenaformis DSM 5348T (89.6%). The average nucleotide identity values between strain SG0102T and related species, K. vitulina DSM 20405T, C. mitsuokai KCTC 5053T, S. azabuensis KCTC 15217T, and E. catenaformis DSM 5348T, were 71.0, 69.3, 70.0, and 69.2%, respectively. The phylogenetic analysis based on 16S rRNA gene sequence revealed that strain SG0102T belonged to the family Erysipelotrichaceae in the class Erysipelotrichia. The DNA G + C content of the strain SG0102T was 39.5 mol%. The major cellular fatty acids (> 10%) of strain SG0102T were C16:0, C16:0 dimethyl acetal, and C18:2?9/12c. The cell wall peptidoglycan of strain SG0102T contained the meso-diaminopimelic acid. The strain SG0102T produced lactic acid as a major end product of fermentation. These distinct phenotypic and phylogenetic properties suggest that strain SG0102T represents a novel species in a novel genus of the family Erysipelotrichaceae, for which the name Intestinibaculum porci gen. nov. sp. nov. is proposed. The type strain is SG0102T (= KCTC 15725T = NBRC 113396T).

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April 21, 2020

Transcriptomic response of Escherichia coli O157 isolates on meat: Comparison between a typical Australian isolate from cattle and a pathogenic clinical isolate

The majority of foodborne illnesses associated with E. coli O157 are attributed to the consumption of foods of bovine origin. In this study, RNA-Seq experiments were undertaken with E. coli O157 to identify genes that may be associated with growth and survival on meat and the beef carcass at low temperature. In addition, the response of an E. coli O157 isolate representative of the general genetic ‘type’ found in Australia (E. coli O157:H- strain EC2422) was compared to that of a pathogenic clinical isolate (E. coli O157:H7 strain Sakai) not typically found in Australia. Both strains up-regulated genes involved in the acid stress response, cold shock response, quorum sensing, biofilm formation and Shiga toxin production. Differences were also observed, with E. coli O157:H7 Sakai up-regulating genes playing a critical role in the barrier function of the outer membrane, lipopolysaccharide biosynthesis, extracellular polysaccharide synthesis and curli production. In contrast, E. coli O157:H- EC2422 down-regulated genes involved in peptidoglycan biosynthesis and of the primary envelope stress response Cpx system. The unique gene expression profiles of the strains, indicate that these genotypes may differ in their ability to persist in the meat production environment and therefore also in their ability to cause disease.

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