scholarly journals Disparate Independent Genetic Events Disrupt the Secondary Metabolism GeneperAin Certain Symbiotic Epichloë Species

2015 ◽  
Vol 81 (8) ◽  
pp. 2797-2807 ◽  
Author(s):  
Daniel Berry ◽  
Johanna E. Takach ◽  
Christopher L. Schardl ◽  
Nikki D. Charlton ◽  
Barry Scott ◽  
...  
Keyword(s):  
Null Alleles ◽  
Polymorphism Analysis ◽  
Symbiotic Association ◽  
Product Size ◽  
Content Type ◽  
Pcr Product ◽  
Peptide Synthetase ◽  
Insertion Mutations ◽  
Reductase Domain ◽  
Prevalent Mutation

ABSTRACTPeramine is an insect-feeding deterrent produced byEpichloëspecies in symbiotic association with C3grasses. TheperAgene responsible for peramine synthesis encodes a two-module nonribosomal peptide synthetase. Alleles ofperAare found in mostEpichloëspecies; however, peramine is not produced by manyperA-containingEpichloëisolates. The genetic basis of these peramine-negative chemotypes is often unknown. Using PCR and DNA sequencing, we analyzed theperAgenes from 72Epichloëisolates and identified causative mutations ofperAnull alleles. We found nonfunctionalperA-ΔR* alleles, which contain a transposon-associated deletion of theperAregion encoding the C-terminal reductase domain, are widespread within theEpichloëgenus and represent a prevalent mutation found in nonhybrid species. Disparate phylogenies of adjacent A2 and T2 domains indicated that the deletion of the reductase domain (R*) likely occurred once and early in the evolution of the genus, and subsequently there have been several recombinations between those domains. A number of novel point, deletion, and insertion mutations responsible for abolishing peramine production in full-lengthperAalleles were also identified. The regions encoding the first and second adenylation domains (A1 and A2, respectively) were common sites for such mutations. Using this information, a method was developed to predict peramine chemotypes by combining PCR product size polymorphism analysis with sequencing of theperAadenylation domains.

scholarly journals Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

2014 ◽  
Vol 80 (7) ◽  
pp. 2125-2132 ◽  
Author(s):  
Narjol Gonzalez-Escalona ◽  
Ruth Timme ◽  
Brian H. Raphael ◽  
Donald Zink ◽  
Shashi K. Sharma
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Clostridium Botulinum ◽  
Toxin Gene ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Group I

ABSTRACTClostridium botulinumis a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA+OrfX−) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA−OrfX+) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producingC. botulinumstrains: two strains with the HA+OrfX−cluster (69A and 32A) and one strain with the HA−OrfX+cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly availableC. botulinumgroup I strains revealed five distinct lineages. Strains 69A and 32A clustered with theC. botulinumtype A1 Hall group, and strain CDC297 clustered with theC. botulinumtype Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination ofC. botulinumgroup I strains and demonstrates the utility of this analysis in quickly differentiatingC. botulinumstrains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.

scholarly journals Specificity of Associations between Bacteria and the Coral Pocillopora meandrina during Early Development

2012 ◽  
Vol 78 (20) ◽  
pp. 7467-7475 ◽  
Author(s):  
Amy Apprill ◽  
Heather Q. Marlow ◽  
Mark Q. Martindale ◽  
Michael S. Rappé
Keyword(s):  
Small Subunit ◽  
Rrna Genes ◽  
Polymorphism Analysis ◽  
Roseobacter Clade ◽  
Small Subunit Rrna ◽  
Content Type ◽  
Sterile Seawater ◽  
Show Evidence ◽  
Coral Health

ABSTRACTRelationships between corals and specific bacterial associates are thought to play an important role in coral health. In this study, the specificity of bacteria associating with the coralPocillopora meandrinawas investigated by exposing coral embryos to various strains of cultured marine bacteria, sterile seawater, or raw seawater and examining the identity, density, and location of incorporated cells. The isolates utilized in this experiment included members of the Roseobacter and SAR11 clades of theAlphaproteobacteria, aPseudoalteromonasspecies of theGammaproteobacteria, and aSynechococcusspecies of theCyanobacteriaphylum. Based on terminal restriction fragment length polymorphism analysis of small-subunit rRNA genes, similarities in bacterial communities associated with 170-h-old planulae were observed regardless of treatment, suggesting that bacteria may have been externally associated from the outset of the experiment. Microscopic examination ofP. meandrinaplanulae by fluorescencein situhybridization with bacterial and Roseobacter clade-specific oligonucleotide probes revealed differences in the densities and locations of planulae-associated cells. Planulae exposed to either raw seawater or strains ofPseudoalteromonasand Roseobacter harbored the highest densities of internally associated cells, of which 20 to 100% belonged to the Roseobacter clade. Planulae exposed to sterile seawater or strains of the SAR11 clade andSynechococcusdid not show evidence of prominent bacterial associations. Additional analysis of the raw-seawater-exposed planulae via electron microscopy confirmed the presence of internally associated prokaryotic cells, as well as virus-like particles. These results suggest that the availability of specific microorganisms may be an important factor in the establishment of coral-bacterial relationships.

scholarly journals Total Biosynthesis and Diverse Applications of the Nonribosomal Peptide-Polyketide Siderophore Yersiniabactin

2015 ◽  
Vol 81 (16) ◽  
pp. 5290-5298 ◽  
Author(s):  
Mahmoud Kamal Ahmadi ◽  
Samar Fawaz ◽  
Charles H. Jones ◽  
Guojian Zhang ◽  
Blaine A. Pfeifer
Keyword(s):  
Polyketide Synthase ◽  
Parallel Applications ◽  
Total Removal ◽  
Heterologous Host ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Metal Chelating ◽  
Removal Capacity ◽  
Peptide Synthetase ◽  
Host Infection

ABSTRACTYersiniabactin (Ybt) is a mixed nonribosomal peptide-polyketide natural product natively produced by the pathogenYersinia pestis. The compound enables iron scavenging capabilities upon host infection and is biosynthesized by a nonribosomal peptide synthetase featuring a polyketide synthase module. This pathway has been engineered for expression and biosynthesis usingEscherichia colias a heterologous host. In the current work, the biosynthetic process for Ybt formation was improved through the incorporation of a dedicated step to eliminate the need for exogenous salicylate provision. When this improvement was made, the compound was tested in parallel applications that highlight the metal-chelating nature of the compound. In the first application, Ybt was assessed as a rust remover, demonstrating a capacity of ∼40% compared to a commercial removal agent and ∼20% relative to total removal capacity. The second application tested Ybt in removing copper from a variety of nonbiological and biological solution mixtures. Success across a variety of media indicates potential utility in diverse scenarios that include environmental and biomedical settings.

scholarly journals Clonal Lineages of Erysipelothrix rhusiopathiae Responsible for Acute Swine Erysipelas in Japan Identified by Using Genome-Wide Single-Nucleotide Polymorphism Analysis

2017 ◽  
Vol 83 (11) ◽  
Author(s):  
Yohsuke Ogawa ◽  
Kazumasa Shiraiwa ◽  
Yoshitoshi Ogura ◽  
Tadasuke Ooka ◽  
Sayaka Nishikawa ◽  
...  
Keyword(s):  
Polymorphism Analysis ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Erysipelothrix Rhusiopathiae ◽  
Single Nucleotide ◽  
Content Type ◽  
Genome Wide ◽  
Wide Range ◽  
Swine Erysipelas ◽  
Lineage Iv

ABSTRACTErysipelothrix rhusiopathiaecauses swine erysipelas, an important infectious disease in the swine industry. In Japan, the incidence of acute swine erysipelas due toE. rhusiopathiaeserovar 1a has recently increased markedly. To study the genetic relatedness of the strains from the recent cases, we analyzed 34E. rhusiopathiaeserovar 1a swine isolates collected between 1990 and 2011 and further investigated the possible association of the live Koganei 65-0.15 vaccine strain (serovar 1a) with the increase in cases. Pulsed-field gel electrophoresis analysis revealed no marked variation among the isolates; however, sequencing analysis of a hypervariable region in the surface-protective antigen A gene (spaA) revealed that the strains isolated after 2007 exhibited the samespaAgenotype and could be differentiated from older strains. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms (SNPs) revealed that the Japanese strains examined were closely related, showing a relatively small number of SNPs among them. The strains were classified into four major lineages, with Koganei 65-0.15 (lineage III) being phylogenetically separated from the other three lineages. The strains isolated after 2007 and the two older strains constituted one major lineage (lineage IV) with a specificspaAgenotype (M203/I257-SpaA), while the recent isolates were further divided into two geographic groups. The remaining older isolates belonged to either lineage I, with the I203/L257-SpaA type, or lineage II, with the I203/I257-SpaA type. These results indicate that the recent increased incidence of acute swine erysipelas in Japan is associated with two sublineages of lineage IV, which have independently evolved in two different geographic regions.IMPORTANCEUsing large-scale whole-genome sequence data fromErysipelothrix rhusiopathiaeisolates from a wide range of hosts and geographic origins, a recent study clarified the existence of three distinct clades (clades 1, 2, and 3) that are found across multiple continents and host species, representing both livestock and wildlife, and an “intermediate” clade between clade 2 and the dominant clade 3 within the species. In this study, we found that theE. rhusiopathiaeJapanese strains examined exhibited remarkably low levels of genetic diversity and confirmed that all of the Japanese and Chinese swine isolates examined in this study belong to clonal lineages within the intermediate clade. We report thatspaAgenotyping ofE. rhusiopathiaestrains is a practical alternative to whole-genome sequencing analysis of theE. rhusiopathiaeisolates from eastern Asian countries.

scholarly journals VibA, a Homologue of a Transcription Factor for Fungal Heterokaryon Incompatibility, Is Involved in Antifungal Compound Production in the Plant-Symbiotic Fungus Epichloë festucae

2014 ◽  
Vol 14 (1) ◽  
pp. 13-24 ◽  
Author(s):  
Jennifer T. Niones ◽  
Daigo Takemoto
Keyword(s):  
Transcription Factor ◽  
Antifungal Activity ◽  
Critical Role ◽  
Antifungal Compound ◽  
Symbiotic Association ◽  
Wild Type ◽  
Exogenous Dna ◽  
Content Type ◽  
Type Isolate ◽  
Epichloë Festucae

ABSTRACT Symbiotic association of epichloae endophytes ( Epichloë/Neotyphodium species) with cool-season grasses of the subfamily Pooideae confers bioprotective benefits to the host plants against abiotic and biotic stresses. While the production of fungal bioprotective metabolites is a well-studied mechanism of host protection from insect herbivory, little is known about the antibiosis mechanism against grass pathogens by the mutualistic endophyte. In this study, an Epichloë festucae mutant defective in antimicrobial substance production was isolated by a mutagenesis approach. In an isolated mutant that had lost antifungal activity, the exogenous DNA fragment was integrated into the promoter region of the vibA gene, encoding a homologue of the transcription factor VIB-1. VIB-1 in Neurospora crassa is a regulator of genes essential in vegetative incompatibility and promotion of cell death. Here we show that deletion of the vibA gene severely affected the antifungal activity of the mutant against the test pathogen Drechslera erythrospila . Further analyses showed that overexpressing vibA enhanced the antifungal activity of the wild-type isolate against test pathogens. Transformants overexpressing vibA showed an inhibitory activity on test pathogens that the wild-type isolate could not. Moreover, overexpressing vibA in a nonantifungal E. festucae wild-type Fl1 isolate enabled the transformant to inhibit the mycelial and spore germination of D. erythrospila . These results demonstrate that enhanced expression of vibA is sufficient for a nonantifungal isolate to obtain antifungal activity, implicating the critical role of VibA in antifungal compound production by epichloae endophytes.

scholarly journals PCR-Based Method forShigella flexneriSerotyping: International Multicenter Validation

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Silvina P. Brengi ◽  
Qiangzheng Sun ◽  
Hilda Bolaños ◽  
Francisco Duarte ◽  
Claire Jenkins ◽  
...  
Keyword(s):  
Traditional Method ◽  
Diarrheal Disease ◽  
Collaborative Work ◽  
Shigella Flexneri ◽  
Culture Collections ◽  
Content Type ◽  
Pcr Product ◽  
Serological Typing ◽  
Multiple Samples ◽  
Multicenter Validation

ABSTRACTShigellaspp. are a leading cause of human diarrheal disease worldwide, withShigella flexneribeing the most frequently isolated species in developing countries. This serogroup is presently classified into 19 serotypes worldwide. We report here a multicenter validation of a multiplex-PCR-based strategy previously developed by Q. Sun, R. Lan, Y. Wang, A. Zhao, et al. (J Clin Microbiol 49:3766–3770, 2011) for molecular serotyping ofS. flexneri. This study was performed by seven international laboratories, with a panel of 71 strains (researchers were blind to their identity) as well as 279 strains collected from each laboratory’s own local culture collections. This collaborative work found a high extent of agreement among laboratories, calculated through interrater reliability (IRR) measures for the PCR test that proved its robustness. Agreement with the traditional method (serology) was also observed in all laboratories for 14 serotypes studied, while specific genetic events could be responsible for the discrepancies among methodologies in the other 5 serotypes, as determined by PCR product sequencing in most of the cases. This work provided an empirical framework that allowed the use of this molecular method to serotypeS. flexneriand showed several advantages over the traditional method of serological typing. These advantages included overcoming the problem of availability of suitable antisera in testing laboratories as well as facilitating the analysis of multiple samples at the same time. The method is also less time-consuming for completion and easier to implement in routine laboratories. We recommend that this PCR be adopted, as it is a reliable diagnostic and characterization methodology that can be used globally for laboratory-based shigella surveillance.

scholarly journals Specific Midgut Region Controlling the Symbiont Population in an Insect-Microbe Gut Symbiotic Association

2013 ◽  
Vol 79 (23) ◽  
pp. 7229-7233 ◽  
Author(s):  
Jiyeun Kate Kim ◽  
Na Hyang Kim ◽  
Ho Am Jang ◽  
Yoshitomo Kikuchi ◽  
Chan-Hee Kim ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Molecular Basis ◽  
Critical Role ◽  
Symbiotic Bacteria ◽  
Symbiotic Association ◽  
Control Mechanisms ◽  
Content Type ◽  
Bean Bug ◽  
Gut Symbionts ◽  
Series Of Experiments

ABSTRACTMany insects possess symbiotic bacteria that affect the biology of the host. The level of the symbiont population in the host is a pivotal factor that modulates the biological outcome of the symbiotic association. Hence, the symbiont population should be maintained at a proper level by the host's control mechanisms. Several mechanisms for controlling intracellular symbionts of insects have been reported, while mechanisms for controlling extracellular gut symbionts of insects are poorly understood. The bean bugRiptortus pedestrisharbors a betaproteobacterial extracellular symbiont of the genusBurkholderiain the midgut symbiotic organ designated the M4 region. We found that the M4B region, which is directly connected to the M4 region, also harborsBurkholderiasymbiont cells, but the symbionts therein are mostly dead. A series of experiments demonstrated that the M4B region exhibits antimicrobial activity, and the antimicrobial activity is specifically potent against theBurkholderiasymbiont but not the culturedBurkholderiaand other bacteria. The antimicrobial activity of the M4B region was detected in symbiotic host insects, reaching its highest point at the fifth instar, but not in aposymbiotic host insects, which suggests the possibility of symbiont-mediated induction of the antimicrobial activity. This antimicrobial activity was not associated with upregulation of antimicrobial peptides of the host. Based on these results, we propose that the M4B region is a specialized gut region ofR. pedestristhat plays a critical role in controlling the population of theBurkholderiagut symbiont. The molecular basis of the antimicrobial activity is of great interest and deserves future study.

scholarly journals Molecular Drug Susceptibility Testing and Genotyping of Mycobacterium leprae Strains from South America

2011 ◽  
Vol 55 (6) ◽  
pp. 2971-2973 ◽  
Author(s):  
Pushpendra Singh ◽  
Philippe Busso ◽  
Alberto Paniz-Mondolfi ◽  
Nacarid Aranzazu ◽  
Marc Monot ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Resistance Mutation ◽  
Mycobacterium Leprae ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Polymorphism Analysis ◽  
South American ◽  
Resistance Mutations ◽  
Single Nucleotide ◽  
Content Type

ABSTRACTPossible drug resistance inMycobacterium lepraestrains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

scholarly journals Inactivation of the Major Hemolysin Gene Influences Expression of the Nonribosomal Peptide Synthetase Gene swrA in the Insect Pathogen Serratia sp. Strain SCBI

2017 ◽  
Vol 199 (21) ◽  
Author(s):  
Lauren M. Petersen ◽  
Kaitlyn LaCourse ◽  
Tim A. Schöner ◽  
Helge Bode ◽  
Louis S. Tisa
Keyword(s):  
Antimicrobial Activity ◽  
Serratia Marcescens ◽  
Virulence Factors ◽  
Nonribosomal Peptide Synthetase ◽  
Mrna Levels ◽  
Insect Pathogen ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Hemolysin Gene ◽  
Peptide Synthetase

ABSTRACT Hemolysins are important virulence factors for many bacterial pathogens, including Serratia marcescens. The role of the major hemolysin gene in the insect pathogen Serratia sp. strain SCBI was investigated using both forward and reverse-genetics approaches. Introduction of the major hemolysin gene into Escherichia coli resulted in a gain of both virulence and hemolytic activity. Inactivation of this hemolysin in Serratia sp. SCBI resulted in a loss of hemolysis but did not attenuate insecticidal activity. Unexpectedly, inactivation of the hemolysin gene in Serratia sp. SCBI resulted in significantly increased motility and increased antimicrobial activity. Reverse transcription-quantitative PCR (qRT-PCR) analysis of mutants with a disrupted hemolysin gene showed a dramatic increase in mRNA levels of a nonribosomal peptide synthetase gene, swrA, which produces the surfactant serrawettin W2. Mutation of the swrA gene in Serratia sp. SCBI resulted in highly varied antibiotic activity, motility, virulence, and hemolysis phenotypes that were dependent on the site of disruption within this 17.75-kb gene. When introduced into E. coli, swrA increases rates of motility and confers antimicrobial activity. While it is unclear how inactivation of the major hemolysin gene influences the expression of swrA, these results suggest that swrA plays an important role in motility and antimicrobial activity in Serratia sp. SCBI. IMPORTANCE The opportunistic Gram-negative bacteria of the genus Serratia are widespread in the environment and can cause human illness. A comparative genomics analysis between Serratia marcescens and a new Serratia species from South Africa, termed Serratia sp. strain SCBI, shows that these two organisms are closely related but differ in pathogenesis. S. marcescens kills Caenorhabditis nematodes, while Serratia sp. SCBI is not harmful and forms a beneficial association with them. This distinction presented the opportunity to investigate potential differences in regulation of common virulence mechanisms between these two species. With the emergence of antibiotic-resistant microorganisms, there is a widespread need to understand the regulation of pathogenesis. The significance of this study is the presentation of evidence for cross-pathway regulation of virulence factors and how the elimination of one mechanism may be compensated for by the upregulation of others.

scholarly journals Culture-Based Methods and Molecular Tools for Azole-Resistant Aspergillus fumigatus Detection in a Belgian University Hospital

2017 ◽  
Vol 55 (8) ◽  
pp. 2391-2399 ◽  
Author(s):  
I. Montesinos ◽  
M. A. Argudín ◽  
M. Hites ◽  
F. Ahajjam ◽  
M. Dodémont ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Lavage Fluid ◽  
University Hospital ◽  
Azole Resistance ◽  
Content Type ◽  
Routine Surveillance ◽  
Improve Patient Management ◽  
Detection Of Mutations ◽  
Improve Patient ◽  
Prevalent Mutation

ABSTRACT Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus ( n = 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR ( n = 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR 34 /L98H was the most prevalent mutation (46.7%), followed by TR 46 /Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology.

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