scholarly journals Characterization of the Bacterial Community of the Chemically Defended Hawaiian Sacoglossan Elysia rufescens

2013 ◽  
Vol 79 (22) ◽  
pp. 7073-7081 ◽  
Author(s):  
Jeanette Davis ◽  
W. Florian Fricke ◽  
Mark T. Hamann ◽  
Eduardo Esquenazi ◽  
Pieter C. Dorrestein ◽  
...  
Keyword(s):  
Longitudinal Section ◽  
Epifluorescence Microscopy ◽  
Ionization Mass ◽  
Bacterial Assemblage ◽  
Content Type ◽  
Anticancer Properties ◽  
Kahalalide F ◽  
Culture Independent ◽  
Elysia Rufescens

ABSTRACTSacoglossans are characterized by the ability to sequester functional chloroplasts from their algal diet through a process called kleptoplasty, enabling them to photosynthesize. The bacterial diversity associated with sacoglossans is not well understood. In this study, we coupled traditional cultivation-based methods with 454 pyrosequencing to examine the bacterial communities of the chemically defended Hawaiian sacoglossanElysia rufescensand its secreted mucus.E. rufescenscontains a defense molecule, kahalalide F, that is possibly of bacterial origin and is of interest because of its antifungal and anticancer properties. Our results showed that there is a diverse bacterial assemblage associated withE. rufescensand its mucus, with secreted mucus harboring higher bacterial richness than entire-E. rufescenssamples. The most-abundant bacterial groups affiliated withE. rufescensand its mucus areMycoplasmaspp. andVibriospp., respectively. Our analyses revealed that theVibriospp. that were highly represented in the cultivable assemblage were also abundant in the culture-independent community. Epifluorescence microscopy and matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS) were utilized to detect the chemical defense molecule kahalalide F on a longitudinal section of the sacoglossan.

scholarly journals Acidianus Tailed Spindle Virus: a New Archaeal Large Tailed Spindle Virus Discovered by Culture-Independent Methods

2016 ◽  
Vol 90 (7) ◽  
pp. 3458-3468 ◽  
Author(s):  
Rebecca A. Hochstein ◽  
Maximiliano J. Amenabar ◽  
Jacob H. Munson-McGee ◽  
Eric S. Boyd ◽  
Mark J. Young
Keyword(s):  
Environmental Samples ◽  
Hot Spring ◽  
Metagenomic Data ◽  
Virus Discovery ◽  
Content Type ◽  
Isolation And Characterization ◽  
Virus Diversity ◽  
Culture Independent ◽  
Domains Of Life

ABSTRACTThe field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea,Bacteria, andEukarya). Traditionally, viral metagenomic studies provide information about viral gene content but rarely provide knowledge about virion morphology and/or cellular host identity. Here we describe a new virus,Acidianustailed spindle virus (ATSV), initially identified by bioinformatic analysis of viral metagenomic data sets from a high-temperature (80°C) acidic (pH 2) hot spring located in Yellowstone National Park, followed by more detailed characterization using only environmental samples without dependency on culturing. Characterization included the identification of the large tailed spindle virion morphology, determination of the complete 70.8-kb circular double-stranded DNA (dsDNA) viral genome content, and identification of its cellular host. Annotation of the ATSV genome revealed a potential three-domain gene product containing an N-terminal leucine-rich repeat domain, followed by a likely posttranslation regulatory region consisting of high serine and threonine content, and a C-terminal ESCRT-III domain, suggesting interplay with the host ESCRT system. The host of ATSV, which is most closely related toAcidianus hospitalis, was determined by a combination of analysis of cellular clustered regularly interspaced short palindromic repeat (CRISPR)/Cas loci and dual viral and cellular fluorescencein situhybridization (viral FISH) analysis of environmental samples and confirmed by culture-based infection studies. This work provides an expanded pathway for the discovery, isolation, and characterization of new viruses using culture-independent approaches and provides a platform for predicting and confirming virus hosts.IMPORTANCEVirus discovery and characterization have been traditionally accomplished by using culture-based methods. While a valuable approach, it is limited by the availability of culturable hosts. In this research, we report a virus-centered approach to virus discovery and characterization, linking viral metagenomic sequences to a virus particle, its sequenced genome, and its host directly in environmental samples, without using culture-dependent methods. This approach provides a pathway for the discovery, isolation, and characterization of new viruses. While this study used an acidic hot spring environment to characterize a new archaeal virus,Acidianustailed spindle virus (ATSV), the approach can be generally applied to any environment to expand knowledge of virus diversity in all three domains of life.

scholarly journals Functional Cloning and Characterization of Antibiotic Resistance Genes from the Chicken Gut Microbiome

2012 ◽  
Vol 78 (8) ◽  
pp. 3028-3032 ◽  
Author(s):  
Wei Zhou ◽  
Ying Wang ◽  
Jun Lin
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Content Type ◽  
Genomic Libraries ◽  
Spectinomycin Resistance ◽  
Culture Independent ◽  
Distant Relative ◽  
Unique Genes

ABSTRACTCulture-independent sampling in conjunction with a functional cloning approach identified diverse antibiotic resistance genes for different classes of antibiotics in gut microbiomes from both conventionally raised and free-range chickens. Many of the genes are phylogenetically distant from known resistance genes. Two unique genes that conferred ampicillin and spectinomycin resistance were also functional inCampylobacter, a distant relative of theEscherichia colihost used to generate the genomic libraries.

scholarly journals Molecular and phenetic characterization of the bacterial assemblage of Hot Lake, WA, an environment with high concentrations of magnesium sulphate, and its relevance to Mars

2014 ◽  
Vol 13 (1) ◽  
pp. 69-80 ◽  
Author(s):  
Brian R. Kilmer ◽  
Timothy C. Eberl ◽  
Brent Cunderla ◽  
Fei Chen ◽  
Benton C. Clark ◽  
...  
Keyword(s):  
Community Analysis ◽  
Initial Study ◽  
Rrna Gene ◽  
Gram Positive ◽  
Bacterial Assemblage ◽  
Planetary Protection ◽  
Culture Independent ◽  
Life Detection ◽  
High Concentrations

AbstractHot Lake (Oroville, WA) is an athalassohaline epsomite lake that can have precipitating concentrations of MgSO4salts, mainly epsomite. Little biotic study has been done on epsomite lakes and it was unclear whether microbes isolated from epsomite lakes and their margins would fall within recognized halotolerant genera, common soil genera or novel phyla. Our initial study cultivated and characterized epsotolerant bacteria from the lake and its margins. Approximately 100 aerobic heterotrophic microbial isolates were obtained by repetitive streak-plating in high-salt media including either 10% NaCl or 2 M MgSO4. The collected isolates were all bacteria, nearly evenly divided between Gram-positive and Gram-negative clades, the most abundant genera beingHalomonas, Idiomarina, Marinobacter, Marinococcus, Nesterenkonia, NocardiopsisandPlanococcus. Bacillus, Corynebacterium, Exiguobacterium, KocuriaandStaphylococcusalso were cultured. This initial study included culture-independent community analysis of direct DNA extracts of lake margin soil using PCR-based clone libraries and 16S rRNA gene phylogeny. Clones assigned to Gram-positive bacterial clades (70% of total clones) were dominated by sequences related to uncultured actinobacteria. There were abundantDeltaproteobacteriaclones related to bacterial sulphur metabolisms and clones ofLegionellaandCoxiella. These epsomite lake microbial communities seem to be divided between bacteria primarily associated with hyperhaline environments rich in NaCl and salinotolerant relatives of common soil organisms. Archaea appear to be in low abundance and none were isolated, despite near-saturated salinities. Growth of microbes at very high concentrations of magnesium and other sulphates has relevance to planetary protection and life-detection missions to Mars, where scant liquid water may form as deliquescent brines and appear as eutectic liquids.

scholarly journals A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Martina Andberg ◽  
Filip Mollerup ◽  
Kirsti Parikka ◽  
Sanna Koutaniemi ◽  
Harry Boer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Uronic Acid ◽  
Modular Structure ◽  
Substrate Preference ◽  
Galactose Oxidase ◽  
Ionization Mass ◽  
Colletotrichum Graminicola ◽  
Content Type ◽  
Identification And Characterization

ABSTRACT We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.−) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family. IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola. As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing.

scholarly journals Evidence for Phytoremediation and Phytoexcretion of NTO from Industrial Wastewater by Vetiver Grass

Molecules ◽  
2020 ◽  
Vol 26 (1) ◽  
pp. 74
Author(s):  
Abhishek RoyChowdhury ◽  
Pallabi Mukherjee ◽  
Saumik Panja ◽  
Rupali Datta ◽  
Christos Christodoulatos ◽  
...  
Keyword(s):  
Stress Reduction ◽  
Industrial Wastewater ◽  
Plant Biomass ◽  
Ionization Mass ◽  
Vetiver Grass ◽  
Insensitive Munitions ◽  
Treatment Technologies ◽  
And Control ◽  
Near Future

The use of insensitive munitions such as 3-nitro-1,2,4-triazol-5-one (NTO) is rapidly increasing and is expected to replace conventional munitions in the near future. Various NTO treatment technologies are being developed for the treatment of wastewater from industrial munition facilities. This is the first study to explore the potential phytoremediation of industrial NTO-wastewater using vetiver grass (Chrysopogon zizanioides L.). Here, we present evidence that vetiver can effectively remove NTO from wastewater, and also translocated NTO from root to shoot. NTO was phytotoxic and resulted in a loss of plant biomass and chlorophyll. The metabolomic analysis showed significant differences between treated and control samples, with the upregulation of specific pathways such as glycerophosphate metabolism and amino acid metabolism, providing a glimpse into the stress alleviation strategy of vetiver. One of the mechanisms of NTO stress reduction was the excretion of solid crystals. Scanning electron microscopy (SEM), electrospray ionization mass spectrometry (ESI-MS), and Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the presence of NTO crystals in the plant exudates. Further characterization of the exudates is in progress to ascertain the purity of these crystals, and if vetiver could be used for phytomining NTO from industrial wastewater.

scholarly journals Characterization of RarA, a Novel AraC Family Multidrug Resistance Regulator in Klebsiella pneumoniae

2012 ◽  
Vol 56 (8) ◽  
pp. 4450-4458 ◽  
Author(s):  
Mark Veleba ◽  
Paul G. Higgins ◽  
Gerardo Gonzalez ◽  
Harald Seifert ◽  
Thamarai Schneiders
Keyword(s):  
Multidrug Resistance ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Content Type ◽  
Resistance Phenotype ◽  
Serratia Proteamaculans ◽  
Virulence Potential ◽  
Article Type ◽  
Acrab Efflux Pump

ABSTRACTTranscriptional regulators, such as SoxS, RamA, MarA, and Rob, which upregulate the AcrAB efflux pump, have been shown to be associated with multidrug resistance in clinically relevant Gram-negative bacteria. In addition to the multidrug resistance phenotype, these regulators have also been shown to play a role in the cellular metabolism and possibly the virulence potential of microbial cells. As such, the increased expression of these proteins is likely to cause pleiotropic phenotypes.Klebsiella pneumoniaeis a major nosocomial pathogen which can express the SoxS, MarA, Rob, and RamA proteins, and the accompanying paper shows that the increased transcription oframAis associated with tigecycline resistance (M. Veleba and T. Schneiders, Antimicrob. Agents Chemother. 56:4466–4467, 2012). Bioinformatic analyses of the availableKlebsiellagenome sequences show that an additional AraC-type regulator is encoded chromosomally. In this work, we characterize this novel AraC-type regulator, hereby called RarA (Regulator of antibiotic resistance A), which is encoded inK. pneumoniae,Enterobactersp. 638,Serratia proteamaculans568, andEnterobacter cloacae. We show that the overexpression ofrarAresults in a multidrug resistance phenotype which requires a functional AcrAB efflux pump but is independent of the other AraC regulators. Quantitative real-time PCR experiments show thatrarA(MGH 78578 KPN_02968) and its neighboring efflux pump operonoqxAB(KPN_02969_02970) are consistently upregulated in clinical isolates collected from various geographical locations (Chile, Turkey, and Germany). Our results suggest thatrarAoverexpression upregulates theoqxABefflux pump. Additionally, it appears thatoqxR, encoding a GntR-type regulator adjacent to theoqxABoperon, is able to downregulate the expression of theoqxABefflux pump, where OqxR complementation resulted in reductions to olaquindox MICs.

scholarly journals Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut

Acta Tropica ◽  
2010 ◽  
Vol 115 (3) ◽  
pp. 275-281 ◽  
Author(s):  
Desiely S. Gusmão ◽  
Adão V. Santos ◽  
Danyelle C. Marini ◽  
Mauricio Bacci ◽  
Marília A. Berbert-Molina ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Bacterial Colonization ◽  
Culture Independent ◽  
Culture Dependent

scholarly journals Characterization of Glycoside Hydrolase Family 5 Proteins in Schizosaccharomyces pombe

2010 ◽  
Vol 9 (11) ◽  
pp. 1650-1660 ◽  
Author(s):  
Encarnación Dueñas-Santero ◽  
Ana Belén Martín-Cuadrado ◽  
Thierry Fontaine ◽  
Jean-Paul Latgé ◽  
Francisco del Rey ◽  
...  
Keyword(s):  
Cell Wall ◽  
Schizosaccharomyces Pombe ◽  
Protein Characterization ◽  
Wall Material ◽  
Glycoside Hydrolase Family ◽  
Content Type ◽  
Hydrolysis Of ◽  
Controlled Hydrolysis ◽  
Hydrolase Family

ABSTRACT In yeast, enzymes with β-glucanase activity are thought to be necessary in morphogenetic events that require controlled hydrolysis of the cell wall. Comparison of the sequence of the Saccharomyces cerevisiae exo-β(1,3)-glucanase Exg1 with the Schizosaccharomyces pombe genome allowed the identification of three genes that were named exg1 + (locus SPBC1105.05), exg2 + (SPAC12B10.11), and exg3 + (SPBC2D10.05). The three proteins have different localizations: Exg1 is secreted to the periplasmic space, Exg2 is a membrane protein, and Exg3 is a cytoplasmic protein. Characterization of the biochemical activity of the proteins indicated that Exg1 and Exg3 are active only against β(1,6)-glucans while no activity was detected for Exg2. Interestingly, Exg1 cleaves the glucans with an endohydrolytic mode of action. exg1 + showed periodic expression during the cell cycle, with a maximum coinciding with the septation process, and its expression was dependent on the transcription factor Sep1. The Exg1 protein localizes to the septum region in a pattern that was different from that of the endo-β(1,3)-glucanase Eng1. Overexpression of Exg2 resulted in an increase in cell wall material at the poles and in the septum, but the putative catalytic activity of the protein was not required for this effect.

scholarly journals Single Clinical Isolates from Acute Uncomplicated Urinary Tract Infections Are Representative of Dominant In Situ Populations

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Dana Willner ◽  
Serene Low ◽  
Jason A. Steen ◽  
Narelle George ◽  
Graeme R. Nimmo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Clinical Isolates ◽  
Content Type ◽  
E Coli ◽  
Strain Diversity ◽  
Amplicon Pyrosequencing ◽  
Culture Independent ◽  
Tract Infections

ABSTRACTUrinary tract infections (UTIs) are one of the most commonly acquired bacterial infections in humans, and uropathogenicEscherichia colistrains are responsible for over 80% of all cases. The standard method for identification of uropathogens in clinical laboratories is cultivation, primarily using solid growth media under aerobic conditions, coupled with morphological and biochemical tests of typically a single isolate colony. However, these methods detect only culturable microorganisms, and characterization is phenotypic in nature. Here, we explored the genotypic identity of communities in acute uncomplicated UTIs from 50 individuals by using culture-independent amplicon pyrosequencing and whole-genome and metagenomic shotgun sequencing. Genus-level characterization of the UTI communities was achieved using the 16S rRNA gene (V8 region). Overall UTI community richness was very low in comparison to other human microbiomes. We strain-typedEscherichia-dominated UTIs using amplicon pyrosequencing of the fimbrial adhesin gene,fimH. There were nine highly abundantfimHtypes, and each UTI sample was dominated by a single type. Molecular analysis of the corresponding clinical isolates revealed that in the majority of cases the isolate was representative of the dominant taxon in the community at both the genus and the strain level. Shotgun sequencing was performed on a subset of eightE. coliurine UTI and isolate pairs. The majority of UTI microbial metagenomic sequences mapped to isolate genomes, confirming the results obtained using phylogenetic markers. We conclude that for the majority of acute uncomplicatedE. coli-mediated UTIs, single cultured isolates are diagnostic of the infection.IMPORTANCEIn clinical practice, the diagnosis and treatment of acute uncomplicated urinary tract infection (UTI) are based on analysis of a single bacterial isolate cultured from urine, and it is assumed that this isolate represents the dominant UTI pathogen. However, these methods detect only culturable bacteria, and the existence of multiple pathogens as well as strain diversity within a single infection is not examined. Here, we explored bacteria present in acute uncomplicated UTIs using culture-independent sequence-based methods.Escherichia coliwas the most common organism identified, and analysis ofE. colidominant UTI samples and their paired clinical isolates revealed that in the majority of infections the cultured isolate was representative of the dominant taxon at both the genus and the strain level. Our data demonstrate that in most cases single cultured isolates are diagnostic of UTI and are consistent with the notion of bottlenecks that limit strain diversity during UTI pathogenesis.

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