scholarly journals Thermus and the Pink Discoloration Defect in Cheese

mSystems ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Lisa Quigley ◽  
Daniel J. O’Sullivan ◽  
David Daly ◽  
Orla O’Sullivan ◽  
Zuzana Burdikova ◽  
...  
Keyword(s):  
16S Rrna ◽  
Quantitative Pcr ◽  
Unknown Origin ◽  
Food Microbiology ◽  
Financial Loss ◽  
Content Type ◽  
Generation Sequencing ◽  
New Strategies ◽  
Pink Discoloration

ABSTRACT Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin. A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch’s postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.

scholarly journals Importance of Micromonospora spp. as Colonizers of Cellulose in Freshwater Lakes as Demonstrated by Quantitative Reverse Transcriptase PCR of 16S rRNA

2012 ◽  
Vol 78 (9) ◽  
pp. 3495-3499 ◽  
Author(s):  
Alexandre B. de Menezes ◽  
James E. McDonald ◽  
Heather E. Allison ◽  
Alan J. McCarthy
Keyword(s):  
16S Rrna ◽  
Reverse Transcriptase ◽  
Quantitative Pcr ◽  
Lake Sediment ◽  
Relative Abundance ◽  
Bacterial Communities ◽  
Bacterial Population ◽  
Freshwater Lakes ◽  
Content Type ◽  
Water Columns

ABSTRACTThe relative abundance of micromonosporas in the bacterial communities inhabiting cellulose baits, water columns, and sediments of two freshwater lakes was determined by quantitative PCR (qPCR) of reverse-transcribed 16S rRNA.Micromonosporaspp. were shown to be significant members of the active bacterial population colonizing cellulosic substrates in the lake sediment, and their increased prevalence with greater depth was confirmed by enumeration of CFU.

scholarly journals Quantitative PCR provides a simple and accessible method for quantitative microbiome profiling

2018 ◽  
Author(s):  
Ching Jian ◽  
Panu Luukkonen ◽  
Hannele Yki-Järvinen ◽  
Anne Salonen ◽  
Katri Korpela
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Quantitative Pcr ◽  
Rrna Gene ◽  
Microbial Community Structures ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Bacterial Groups ◽  
Microbiome Profiling ◽  
Generation Sequencing

AbstractThe use of relative next generation sequencing (NGS) abundance data can lead to misinterpretations of microbial community structures as the increase of one taxon leads to concurrent decrease of the other(s). To overcome compositionality, we provide a quantitative NGS solution, which is achieved by adjusting the relative 16S rRNA gene amplicon NGS data with quantitative PCR (qPCR-based) total bacterial counts. By comparing the enumeration of dominant bacterial groups on different taxonomic levels in human fecal samples using taxon-specific 16S rRNA gene-targeted qPCR we show that quantitative NGS is able to estimate absolute bacterial abundances accurately. We also observed a higher degree of correspondence in the estimated microbe-metabolite relationship when quantitative NGS was applied. Being conceptually and methodologically analogous to amplicon-based NGS, our qPCR-based method can be readily incorporated into the standard, high-throughput NGS sample processing pipeline for more accurate description of interactions within and between the microbes and host.

scholarly journals Composition and Origin of the Fermentation Microbiota of Mahewu, a Zimbabwean Fermented Cereal Beverage

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Felicitas Pswarayi ◽  
Michael G. Gänzle
Keyword(s):  
16S Rrna ◽  
Quantitative Pcr ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Culture Methods ◽  
Content Type ◽  
Cell Counts

ABSTRACTMahewu is a fermented cereal beverage produced in Zimbabwe. This study determined the composition and origin of mahewu microbiota. The microbiota of mahewu samples consisted of 3 to 7 dominant strains of lactobacilli and two strains of yeasts.Enterobacteriaceaewere not detected.Candida glabratawas present in high cell counts from samples collected in summer but not from samples collected in winter. Millet malt is the only raw ingredient used in the production of mahewu and is a likely source of fermentation microbiota; therefore, malt microbiota was also analyzed by culture-dependent and high-throughput 16S rRNA gene sequencing methodologies. Millet malt contained 8 to 19 strains ofEnterobacteriaceae, lactobacilli, bacilli, and very few yeasts. Strain-specific quantitative PCR assays were established on the basis of the genome sequences ofLactobacillus fermentumFUA3588 and FUA3589 andLactobacillus plantarumFUA3590 to obtain a direct assessment of the identity of strains from malt and mahewu.L. fermentumFUA3588 and FUA3589 were detected in millet malt, demonstrating that millet malt is a main source of mahewu microbiota. Strains which were detected in summer were not detected in samples produced at the same site in winter. Model mahewu fermentations conducted with a 5-strain inoculum consisting of lactobacilli,Klebsiella pneumoniae,andCronobacter sakazakiidemonstrated that lactobacilli outcompeteEnterobacteriaceae, which sharply decreased in the first 24 h. In conclusion, mahewu microbiota is mainly derived from millet malt microbiota, but minor components of malt microbiota rapidly outcompeteEnterobacteriaceaeandBacillusspecies during fermentation.IMPORTANCEThis study provides insight into the composition and origin of the microbiota of mahewu and the composition of millet malt microbiota. Fermentation microbiota are often hypothesized to be derived from the environment, but the evidence remains inconclusive. Our findings confirm that millet malt is the major source of mahewu microbiota. By complementing culture methods with high-throughput sequencing of 16S rRNA amplicons and strain-specific quantitative PCR, this study provides evidence about the source of mahewu microbiota, which can inform the development of starter cultures for mahewu production. The study also documents the fate ofEnterobacteriaceaeduring the fermentation of mahewu. There are concerns regarding the safety of traditionally prepared mahewu, and this requires in-depth knowledge of the fermentation process. Therefore, this study elucidated millet malt microbiota and identified cultures that are able to control the high numbers ofEnterobacteriaceaethat are initially present in mahewu fermentations.

scholarly journals Natural Niche for Organohalide-Respiring Chloroflexi

2011 ◽  
Vol 78 (2) ◽  
pp. 393-401 ◽  
Author(s):  
Mark J. Krzmarzick ◽  
Benjamin B. Crary ◽  
Jevon J. Harding ◽  
Oyenike O. Oyerinde ◽  
Alessandra C. Leri ◽  
...  
Keyword(s):  
16S Rrna ◽  
Terrestrial Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Soil Core ◽  
Content Type ◽  
Integral Role ◽  
Terrestrial Environments ◽  
Inorganic Chloride

ABSTRACTThe phylumChloroflexicontains several isolated bacteria that have been found to respire a diverse array of halogenated anthropogenic chemicals. The distribution and role of theseChloroflexiin uncontaminated terrestrial environments, where abundant natural organohalogens could function as potential electron acceptors, have not been studied. Soil samples (116 total, including 6 sectioned cores) from a range of uncontaminated sites were analyzed for the number ofDehalococcoides-likeChloroflexi16S rRNA genes present.Dehalococcoides-likeChloroflexipopulations were detected in all but 13 samples. The concentrations of organochlorine ([organochlorine]), inorganic chloride, and total organic carbon (TOC) were obtained for 67 soil core sections. The number ofDehalococcoides-likeChloroflexi16S rRNA genes positively correlated with [organochlorine]/TOC while the number ofBacteria16S rRNA genes did not.Dehalococcoides-likeChloroflexiwere also observed to increase in number with a concomitant accumulation of chloride when cultured with an enzymatically produced mixture of organochlorines. This research provides evidence that organohalide-respiringChloroflexiare widely distributed as part of uncontaminated terrestrial ecosystems, they are correlated with the fraction of TOC present as organochlorines, and they increase in abundance while dechlorinating organochlorines. These findings suggest that organohalide-respiringChlorofleximay play an integral role in the biogeochemical chlorine cycle.

scholarly journals RNase III Processing of rRNA in the Lyme Disease SpirocheteBorrelia burgdorferi

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Melissa L. Anacker ◽  
Dan Drecktrah ◽  
Richard D. LeCoultre ◽  
Meghan Lybecker ◽  
D. Scott Samuels
Keyword(s):  
Lyme Disease ◽  
16S Rrna ◽  
5S Rrna ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Rnase Iii ◽  
Content Type ◽  
In The Wild

ABSTRACTThe rRNA genes ofBorrelia(Borreliella)burgdorferiare unusually organized; the spirochete has a single 16S rRNA gene that is more than 3 kb from a tandem pair of 23S-5S rRNA operons. We generated anrncnull mutant inB. burgdorferithat exhibits a pleiotropic phenotype, including decreased growth rate and increased cell length. Here, we demonstrate that endoribonuclease III (RNase III) is, as expected, involved in processing the 23S rRNA inB. burgdorferi. The 5′ and 3′ ends of the three rRNAs were determined in the wild type andrncBbmutants; the results suggest that RNase III inB. burgdorferiis required for the full maturation of the 23S rRNA but not for the 5S rRNA nor, curiously, for the 16S rRNA.IMPORTANCELyme disease, the most common tick-borne zoonosis in the Northern Hemisphere, is caused by the bacteriumBorrelia(Borreliella)burgdorferi, a member of the deeply branching spirochete phylum.B. burgdorfericarries a limited suite of ribonucleases, enzymes that cleave RNA during processing and degradation. Several ribonucleases, including RNase III, are involved in the production of ribosomes, which catalyze translation and are a major target of antibiotics. This is the first study to dissect the role of an RNase in any spirochete. We demonstrate that an RNase III mutant is viable but has altered processing of rRNA.

scholarly journals Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

2012 ◽  
Vol 79 (1) ◽  
pp. 196-204 ◽  
Author(s):  
Hodon Ryu ◽  
Michael Henson ◽  
Michael Elk ◽  
Carlos Toledo-Hernandez ◽  
John Griffith ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Quantitative Pcr ◽  
Water Samples ◽  
The Other ◽  
Rrna Gene ◽  
Fecal Samples ◽  
Content Type ◽  
Specific Assay ◽  
Enterococcal Species

ABSTRACTThe detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples wereEnterococcus faecalisandEnterococcus faecium, although we identified more water isolates asEnterococcus casseliflavusthan as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

scholarly journals Functional Role of Methylation of G518 of the 16S rRNA 530 Loop by GidB in Mycobacterium tuberculosis

2013 ◽  
Vol 57 (12) ◽  
pp. 6311-6318 ◽  
Author(s):  
Sharon Y. Wong ◽  
Babak Javid ◽  
Balasubrahmanyam Addepalli ◽  
Grzegorz Piszczek ◽  
Michael Brad Strader ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rrna ◽  
Functional Role ◽  
High Performance ◽  
Protein Translation ◽  
Titration Calorimetry ◽  
Wild Type ◽  
Content Type ◽  
Wobble Position

ABSTRACTPosttranscriptional modifications of bacterial rRNA serve a variety of purposes, from stabilizing ribosome structure to preserving its functional integrity. Here, we investigated the functional role of one rRNA modification in particular—the methylation of guanosine at position 518 (G518) of the 16S rRNA inMycobacterium tuberculosis. Based on previously reported evidence that G518 is located 5 Å; from proline 44 of ribosomal protein S12, which interacts directly with the mRNA wobble position of the codon:anticodon helix at the A site during translation, we speculated that methylation of G518 affects protein translation. We transformed reporter constructs designed to probe the effect of functional lesions at one of the three codon positions on translational fidelity into the wild-type strain, H37Rv, and into a ΔgidBmutant, which lacks the methyltransferase (GidB) that methylates G518. We show that mistranslation occurs less in the ΔgidBmutant only in the construct bearing a lesion in the wobble position compared to H37Rv. Thus, the methylation of G518 allows mistranslation to occur at some level in order for translation to proceed smoothly and efficiently. We also explored the role of methylation at G518 in altering the susceptibility ofM. tuberculosisto streptomycin (SM). Using high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS), we confirmed that G518 is not methylated in the ΔgidBmutant. Furthermore, isothermal titration calorimetry experiments performed on 70S ribosomes purified from wild-type and ΔgidBmutant strains showed that methylation significantly enhances SM binding. These results provide a mechanistic explanation for the low-level, SM-resistant phenotype observed inM. tuberculosisstrains that contain agidBmutation.

Exploring the sources and role of knowledge sharing to overcome the challenges of organizational change implementation

2018 ◽  
Vol 26 (3) ◽  
pp. 567-581 ◽  
Author(s):  
Usman Aslam ◽  
Farwa Muqadas ◽  
Muhammad Kashif Imran ◽  
Ubaid-Ur-Rahman Ubaid-Ur-Rahman
Keyword(s):  
Organizational Change ◽  
Knowledge Sharing ◽  
Change Implementation ◽  
Content Type ◽  
Interview Method ◽  
Social Structural ◽  
Public Sector Organizations ◽  
Change Leaders ◽  
New Strategies

Purpose In recent times, organizations and leaders are focusing on new strategies to increase the success rate of organizational change (OC) implementation. Therefore, this study aims to uncover the sources and role of knowledge sharing (KS) to overcome the challenges of OC implementation. Design/methodology/approach For this research, data have been gathered from respondents based on their key designations by using unstructured interview method. Thematic analysis was then performed using the NVivo 11-Plus software. Findings It has found that employees in public sector organizations are opposing OC because of ineffective communication, and a lower level of employee participation in decision-making and barriers of, for example, a cultural, social, structural and political nature. Therefore, this study suggests how KS can be used to overcome the challenges of OC implementation. Research limitations/implications KS has become necessary to ensure incremental and radical changes in the survival of dynamic businesses. The results are useful to enhance understanding regarding the role of KS in the context of OC among change leaders, researchers, academicians and policymakers. Originality/value The study provides meaningful and novel knowledge regarding role of KS to overcome the challenges of OC implementation. No prior research that contributes practical and theoretical knowledge in the perspective of KS and OC has been found, especially in the context of developing countries and Asian culture. Therefore, this investigation attempts to explore the role of KS and presented overarching conceptual framework in the real context of OC implementation.

scholarly journals Bioinformatics Resources for MicroRNA Discovery

2015 ◽  
Vol 10s4 ◽  
pp. BMI.S29513 ◽  
Author(s):  
Alyssa C. Moore ◽  
Jonathan S. Winkjer ◽  
Tsai-Tien Tseng
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Biomarker Identification ◽  
New Information ◽  
Generation Sequencing ◽  
New Strategies

Biomarker identification is often associated with the diagnosis and evaluation of various diseases. Recently, the role of microRNA (miRNA) has been implicated in the development of diseases, particularly cancer. With the advent of next-generation sequencing, the amount of data on miRNA has increased tremendously in the last decade, requiring new bioinformatics approaches for processing and storing new information. New strategies have been developed in mining these sequencing datasets to allow better understanding toward the actions of miRNAs. As a result, many databases have also been established to disseminate these findings. This review focuses on several curated databases of miRNAs and their targets from both predicted and validated sources.

scholarly journals Capturing Compositional Variation in Denitrifying Communities: a Multiple-Primer Approach That Includes Epsilonproteobacteria

2017 ◽  
Vol 83 (6) ◽  
Author(s):  
Sheryl A. Murdock ◽  
S. Kim Juniper
Keyword(s):  
16S Rrna ◽  
Quantitative Pcr ◽  
Nitrite Reductase ◽  
Nitrogen Loss ◽  
Global Ocean ◽  
Future Application ◽  
Compositional Variation ◽  
Rrna Gene ◽  
Content Type ◽  
Marine Habitats

ABSTRACT Denitrifying Epsilonproteobacteria may dominate nitrogen loss processes in marine habitats with intense redox gradients, but assessment of their importance is limited by the currently available primers for nitrite reductase genes. Nine new primers targeting the nirS gene of denitrifying Epsilonproteobacteria were designed and tested for use in sequencing and quantitative PCR on two microbial mat samples (vent 2 and vent 4) from the Calypso hydrothermal vent field, Bay of Plenty, New Zealand. Commonly used nirS and nirK primer sets nirS1F/nirS6R, cd3aF/R3cd, nirK1F/nirK5R, and F1aCu/R3Cu were also tested to determine what may be missed by the common single-primer approach to assessing denitrifier diversity. The relative importance of Epsilonproteobacteria in these samples was evaluated by 16S rRNA gene sequencing. Epsilonproteobacteria represented up to 75.6% of 16S rRNA libraries, but nirS genes from this group were not found with commonly used primers. Pairing of the new primer EPSnirS511F with either EPSnirS1100R or EPSnirS1105R recovered nirS sequences from members of the genera Sulfurimonas, Sulfurovum, and Nitratifractor. The new quantitative PCR primers EPSnirS103F/EPSnirS530R showed dominance of denitrifying Epsilonproteobacteria in vent 4 compared to vent 2, which had greater representation by “standard” denitrifiers measured with the cd3aF/R3cd primers. Limited results from commonly used nirK primers suggest biased amplification between primers. Future application of multiple nirS and nirK primers, including the new epsilonproteobacterial nirS primers, will improve the detection of denitrifier diversity and the capability to identify changes in dominant denitrifying communities. IMPORTANCE Estimating the potential for increasing nitrogen limitation in the changing global ocean is reliant on understanding the microbial community that removes nitrogen through the process of denitrification. This process is favored under oxygen limitation, which is a growing global-ocean phenomenon. Current methods use the nitrite reductase genes nirS and nirK to assess denitrifier diversity and abundance using primers that target only a few known denitrifiers and systematically exclude denitrifying Epsilonproteobacteria, a group known to dominate in reducing environments, such as hydrothermal vents and anoxic basins. As oxygen depletion expands in the oceans, it is important to study denitrifier community dynamics within those areas to predict future global ocean changes. This study explores the design and testing of new primers that target epsilonproteobacterial nirS and reveals the varied success of existing primers, leading to the recommendation of a multiple-primer approach to assessing denitrifier diversity.

Sign in / Sign up

Export Citation Format

Share Document