scholarly journals Gene Structure and Transcriptional Organization of the dnaK Operon of Bifidobacterium breve UCC 2003 and Application of the Operon in Bifidobacterial Tracing

2005 ◽  
Vol 71 (1) ◽  
pp. 487-500 ◽  
Author(s):  
Marco Ventura ◽  
Ralf Zink ◽  
Gerald F. Fitzgerald ◽  
Douwe van Sinderen
Keyword(s):  
16S Rrna ◽  
Osmotic Shock ◽  
Gene Region ◽  
Evolutionary Development ◽  
Pcr Analysis ◽  
Northern Hybridization ◽  
Gene Marker ◽  
Rrna Gene ◽  
Bifidobacterium Breve ◽  
Polycistronic Mrna

ABSTRACT The incorporation and delivery of bifidobacterial strains as probiotic components in many food preparations expose these microorganisms to a multitude of environmental insults, including heat and osmotic stresses. We characterized the dnaK gene region of Bifidobacterium breve UCC 2003. Sequence analysis of the dnaK locus revealed four genes with the organization dnaK-grpE-dnaJ-ORF1, whose deduced protein products display significant similarity to corresponding chaperones found in other bacteria. Northern hybridization and real-time LightCycler PCR analysis revealed that the transcription of the dnaK operon was strongly induced by osmotic shock but was not induced significantly by heat stress. A 4.4-kb polycistronic mRNA, which represented the transcript of the complete dnaK gene region, was detected. Many other small transcripts, which were assumed to have resulted from intensive processing or degradation of this polycistronic mRNA, were identified. The transcription start site of the dnaK operon was determined by primer extension. Phylogenetic analysis of the available bifidobacterial grpE and dnaK genes suggested that the evolutionary development of these genes has been similar. The phylogeny derived from the various bifidobacterial grpE and dnaK sequences is consistent with that derived from 16S rRNA. The use of these genes in bifidobacterial species as an alternative or complement to the 16S rRNA gene marker provides sequence signatures that allow a high level of discrimination between closely related species of this genus.

scholarly journals Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Christine Drengenes ◽  
Tomas M. L. Eagan ◽  
Ingvild Haaland ◽  
Harald G. Wiker ◽  
Rune Nielsen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Load ◽  
Marker Gene ◽  
Gene Region ◽  
Lower Airway ◽  
Rrna Gene ◽  
Wide Range ◽  
Airway Microbiome ◽  
The Impact

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

scholarly journals REP-PCR Analysis to Study Prokaryotic Biodiversity from Lake Meyghan

2017 ◽  
Vol 61 ◽  
pp. 69-84 ◽  
Author(s):  
Ali Naghoni ◽  
Giti Emtiazi ◽  
Mohammad Ali Amoozegar ◽  
Zahra Etemadifar ◽  
Seyed Abolhassan Shahzadeh Fazeli
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
White Region ◽  
Rrna Gene Sequencing

Repetitive extragenic palindromic elements-polymerase chain reaction (rep-PCR) with 16S ribosomal ribonucleic acid (16S rRNA) genes sequences successfully used for the analysis of microbial community. In this study, the prokaryotic community in Lake Meyghan described by using rep-PCR analysis along with 16S rRNA gene sequencing. The water samples were collected from Lake Meyghan in November 2013. All samples were diluted and cultured on three different media. To estimate the number of prokaryotes per milliliter of the lake we used quantitative real‑time PCR (qPCR). Rep-PCR combination with 16S rRNA gene sequencing was performed to investigate prokaryotes biodiversity in the lake. 305 strains were isolated in this work; 113 isolates for green region, 102 isolates for red region, and 90 isolates for white region. The dendrograms generated 10, 7, and 9 clusters for a 70 % similarity cut-off for green, red, and white regions, respectively. Based on rep-PCR and 16S rRNA gene sequencing, the recovered isolates were dominated by (77.5 %)Halobacteriacaeand many isolates were related to the generaHalorubrum,Haloarcula,Haloterrigena,Natrinema, andHalovivaxin the white region. In the red region more isolated strains (57.5 %) belonged toBacillaceaeand the remaining 42.5 % of isolates belonged to archaea domain,Halorubrum, andHaloarcula. In the green region members ofGammaproteobacteriawere recoverd, this region was dominant withPseudoalteromonas,Salinivibrio, andAliidiomarina.

Streptomyces rhizophilus Causes Potato Common Scab Disease

Plant Disease ◽  
2021 ◽  
Author(s):  
Qi Wei ◽  
Jie Li ◽  
Shuai Yang ◽  
Wenzhong Wang ◽  
Fanxiang Min ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Common Scab ◽  
Bacterial Species ◽  
Economic Losses ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Heilongjiang Province ◽  
Epidemic Disease ◽  
Genus Streptomyces

Common scab (CS) caused by Streptomyces spp. is a significant soilborne potato disease that results in tremendous economic losses globally. Identification of CS-associated species of the genus Streptomyces can enhance understanding of the genetic variation of these bacterial species and is necessary for the control of this epidemic disease. The present study isolated Streptomyces strain 6-2-1(1) from scabby potatoes in Keshan County, Heilongjiang Province, China. PCR analysis confirmed that the strain harbored the characteristic Streptomyces pathogenicity island (PAI) genes (txtA, txtAB, nec1, and tomA). Pathogenicity assays proved that the strain caused typical scab lesions on potato tuber surfaces and necrosis on radish seedlings and potato slices. Subsequently, the strain was systemically characterized at morphological, physiological, biochemical and phylogenetic levels. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 6-2-1(1) shared 99.86% sequence similarity with Streptomyces rhizophilus JR-41T, isolated initially from bamboo in rhizospheric soil in Korea. PCR amplification followed by Sanger sequencing of the 16S rRNA gene of 164 scabby potato samples collected in Heilongjiang Province from 2019 to 2020 demonstrated that approximately 2% of the tested samples were infected with S. rhizophilus. Taken together, these results demonstrate that S. rhizophilus is capable of causing potato CS disease and may pose a potential challenge to potato production in Heilongjiang Province of China.

scholarly journals Ibuprofen Degradation and Associated Bacterial Communities in Hyporheic Zone Sediments

2020 ◽  
Vol 8 (8) ◽  
pp. 1245
Author(s):  
Cyrus Rutere ◽  
Kirsten Knoop ◽  
Malte Posselt ◽  
Adrian Ho ◽  
Marcus A. Horn
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Hyporheic Zone ◽  
Community Dynamics ◽  
Environmental Concern ◽  
Transformation Products ◽  
Pollutant Removal ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Microbial Community Dynamics

Ibuprofen, a non-steroidal anti-inflammatory pain reliever, is among pharmaceutical residues of environmental concern ubiquitously detected in wastewater effluents and receiving rivers. Thus, ibuprofen removal potentials and associated bacteria in the hyporheic zone sediments of an impacted river were investigated. Microbially mediated ibuprofen degradation was determined in oxic sediment microcosms amended with ibuprofen (5, 40, 200, and 400 µM), or ibuprofen and acetate, relative to an un-amended control. Ibuprofen was removed by the original sediment microbial community as well as in ibuprofen-enrichments obtained by re-feeding of ibuprofen. Here, 1-, 2-, 3-hydroxy- and carboxy-ibuprofen were the primary transformation products. Quantitative real-time PCR analysis revealed a significantly higher 16S rRNA abundance in ibuprofen-amended relative to un-amended incubations. Time-resolved microbial community dynamics evaluated by 16S rRNA gene and 16S rRNA analyses revealed many new ibuprofen responsive taxa of the Acidobacteria, Actinobacteria, Bacteroidetes, Gemmatimonadetes, Latescibacteria, and Proteobacteria. Two ibuprofen-degrading strains belonging to the genera Novosphingobium and Pseudomonas were isolated from the ibuprofen-enriched sediments, consuming 400 and 300 µM ibuprofen within three and eight days, respectively. The collective results indicated that the hyporheic zone sediments sustain an efficient biotic (micro-)pollutant degradation potential, and hitherto unknown microbial diversity associated with such (micro)pollutant removal.

scholarly journals Genetic Characterization of the Bifidobacterium breve UCC 2003 hrcA Locus

2005 ◽  
Vol 71 (12) ◽  
pp. 8998-9007 ◽  
Author(s):  
Marco Ventura ◽  
Carlos Canchaya ◽  
Valentina Bernini ◽  
Antonio Del Casale ◽  
Franco Dellaglio ◽  
...  
Keyword(s):  
Genetic Characterization ◽  
Osmotic Shock ◽  
Transcriptional Regulators ◽  
Adaptation Strategy ◽  
Evolutionary Development ◽  
Bifidobacterium Breve ◽  
Induction Pattern ◽  
Shock Response ◽  
Elevated Expression

ABSTRACT The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and transcriptional regulators, including the DnaJ and the HrcA proteins. Genome analysis of Bifidobacterium breve UCC 2003 revealed a second copy of a dnaJ gene, named dnaJ 2, which is flanked by the hrcA gene in a genetic constellation that appears to be unique to the actinobacteria. Phylogenetic analysis using 53 bacterial dnaJ sequences, including both dnaJ 1 and dnaJ 2 sequences, suggests that these genes have followed a different evolutionary development. Furthermore, the B. breve UCC 2003 dnaJ 2 gene seems to be regulated in a manner that is different from that of the previously characterized dnaJ 1 gene. The dnaJ 2 gene, which was shown to be part of a 2.3-kb bicistronic operon with hrcA, was induced by osmotic shock but not significantly by heat stress. This induction pattern is unlike those of other characterized dnaJ genes and may be indicative of a unique stress adaptation strategy by this commensal microorganism.

scholarly journals Molecular Detection and Phylogenetic Analysis of Anaplasma Spp. in Cattle in Al-Qadisiyah Province of Iraq

2019 ◽  
Vol 42 (2) ◽  
pp. 181-188
Author(s):  
Hayder N. Ayyez ◽  
Yahia I. Khudhair ◽  
Qassim Haleem Kshash
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Control Strategies ◽  
Age Groups ◽  
Cattle Breed ◽  
16S Rrna Gene Sequencing ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

AbstractAnaplasma spp. are widely spread rickettsial bacteria transmitted by ticks and placing high impacts on veterinary and public health. A limited number of studies have been carried out on Anaplasmosis in the central part of Iraq. This study was conducted to determine the presence of Anaplasma spp. in cattle in Al-Qadisiyah province, Iraq. A total of 400 blood specimens were collected from cattle suffering from heavy tick infestation. Cattle were blood-sampled from four hyper-endemic areas with ticks. Blood samples were screened using microscopic and polymerase chain reaction (PCR) methods. Diff-quick stained blood smears revealed Anaplasma-like inclusion bodies in 254 (63.5%) samples. According to the 16S rRNA-gene-based PCR analysis, Anaplasma spp. was detected in 124 of the 400 (31%) samples, divided as 96/254 (37.8%) among the microscopical positive samples and 28/146 (19.17%) among the microscopical negative samples. Phylogenetic analysis based on the partial 16S rRNA gene sequencing of ten-PCR positive samples were 99–97% identical to sequences deposited in the GenBank, revealing presence of A. phagocytophilum, A. marginale and unnamed Anaplasma spp. in 40%, 20%, and 40% samples, respectively. Relationships among Anaplasma spp. infections and cattle breed, age, and sex were analyzed. Calves less than one year old showed significantly higher rates (p<0.005) than those from other age groups, whereas sex and breed demonstrated no significant differences (p˃0.001). This study shows that a variety of Anaplasma spp., were endemic in central part of Iraq and is still a hidden problem in cattle in the hyperendemic areas of tick, which requires serious control strategies.

scholarly journals THE 16S rRNA GENE MARKER-BASED MOLECULAR IDENTIFICATION OF CULTURABLE COLIFORM BACTERIA ISOLATED FROM FORAMINIFERA CALCARINA DERIVED FROM PRAMUKA ISLAND WATERS, THE SERIBU ISLAND DISTRICT, JAKARTA PROVINCE

2020 ◽  
Vol 13 (1) ◽  
pp. 10-18
Author(s):  
Mochamad Untung Kurnia - Agung ◽  
Agus Tri Askar ◽  
Yuli Andriani ◽  
Lintang Permatasari Yuliadi
Keyword(s):  
Coral Reef ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Identification ◽  
Bay Of Bengal ◽  
Coliform Bacteria ◽  
Gene Marker ◽  
Rrna Gene ◽  
Gene Markers ◽  
The 16S Rrna Gene

Contamination of coliform bacteria in benthic foraminifera has been reported due to pollution of organic wastes in the aquatic environment around coral reef ecosystems and this event was known to interfere the process of foraminifera shell formation which in turn resulted the disruption of the role of foraminifera in the process of formation of coral reef bottom sediments. The aim of this research is to identify the isolates of culturable coliform bacteria that contaminate foraminifera Calcarina species isolated from the waters of the Pramuka Island, the Seribu Island district, Jakarta Province using the 16S rRNA gene markers. Foraminifera sampling was carried out in the waters of Pramuka Island, the Seribu Island district, Jakarta Province in 5 (five) stations, while the process of bacterial isolation and molecular identification were carried out at the Laboratory of Microbiology and Molecular Biotechnology (MICROMOL), Faculty of Fisheries and Marine Sciences (FPIK), University Padjadjaran. Molecular identification was carried out using the Polymerase Chain Reaction (PCR) method based on the 16S rRNA gene markers. Sequencing is done by sending PCR results to 1st Base, sequencing service company, in Singapore and then, the aligning of sequencing results with databases in genBank was done using  the Basic Local Alignment Search Tool (BLASTTM) program available on the National Center for Biotechnology Information (NCBI) website. The results of 16S rRNA gene amplification from the five isolates produced amplicons of ± 1400 bp length with concentrations ranging from 157.5 µg / mL-230 µg / mL and with a purity ratio ranging from 1.477-1.769. While the results of BLAST and phylogenetic analysis showed that the five isolates were closely related to the isolate Eschericia coli strain inspire99 (Acc No. JQ315935.1), which was isolated from the waters of the Bay of Bengal, India. These results also indicate the existence of ecological connectivity between the waters of the Bay of Bengal in India and the waters of Pramuka Island in Indonesia.

scholarly journals Single-Nucleotide Primer Extension Assay for Detection and Sequence Typing of “Dehalococcoides” spp.

2007 ◽  
Vol 74 (1) ◽  
pp. 300-304 ◽  
Author(s):  
Marcell Nikolausz ◽  
Antonis Chatzinotas ◽  
Márton Palatinszky ◽  
Gwenaël Imfeld ◽  
Paula Martinez ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Primer Extension ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Single Nucleotide ◽  
Specific Pcr ◽  
Novel Approach ◽  
Signature Sequences ◽  
Single Nucleotide Primer Extension

ABSTRACT A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus “Dehalococcoides”. The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.

PSYCHROPHILIC STRAIN FUSARIUM OXYSPORUM – PROSPECTS FOR THE USE FOR BIODEGRADATION OF CYANIDES IN INDUSTRIAL CIRCULATING WATERS

2020 ◽  
pp. 316-318
Author(s):  
Yu. Litovka ◽  
S. Petrenko ◽  
R. Enazarov
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fusarium Oxysporum ◽  
Deep Sequencing ◽  
Gene Region ◽  
Rrna Gene ◽  
Cultivation Conditions ◽  
Submerged Wood ◽  
The 16S Rrna Gene

The microbiome of industrial cyanide-containing circulating waters studied using deep sequencing of the 16S rRNA gene region. Microorganisms are isolated from water, perennial submerged wood and Equisetum fluviatile. Cyanide bio destructors were screened; cultivation conditions of the Fusarium oxysporum strain in a shaker incubator and bioreactor were selected.

Novel mycolic acid-containing bacteria in the family Segniliparaceae fam. nov., including the genus Segniliparus gen. nov., with descriptions of Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov.

2005 ◽  
Vol 55 (4) ◽  
pp. 1615-1624 ◽  
Author(s):  
W. Ray Butler ◽  
Margaret M. Floyd ◽  
June M. Brown ◽  
Sean R. Toney ◽  
Maryam I. Daneshvar ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Health Department ◽  
Mycolic Acid ◽  
Diaminopimelic Acid ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Mycolic Acids

Four strains of novel, rapidly growing, acid–alcohol-fast-staining bacteria were characterized with a polyphasic approach. Isolates were received by the Centers for Disease Control and Prevention from domestic health department laboratories for reference testing as unidentifiable, clinical mycobacteria. Bacteria were rod-shaped and produced non-pigmented (white to beige), non-photochromogenic, smooth or wrinkled-rough colonies on Middlebrook 7H10 and 7H11 media at 33 °C. The smooth and wrinkled colony forms were representative of two species with 68·0 and 72·0 mol% DNA G+C content. The cell wall contained meso-diaminopimelic acid and mycolic acids. Species were characterized by cellular fatty acids of C10 : 0, C14 : 0, C16 : 1ω9t, C16 : 0, C18 : 1ω9c and 10-methyl C18 : 0 (tuberculostearic acid). HPLC analysis of mycolic acids produced a novel late-emerging, genus-specific mycolate pattern. TLC analysis demonstrated a novel α +-mycolate. Species were 98·9 % similar by comparison of 16S rRNA gene sequences; however, the DNA–DNA association was <28 %. Phylogenetic analysis of 16S rRNA gene sequences demonstrated an association with Rhodococcus equi, although a DNA–DNA relatedness value of 2 % did not support a close relationship. PCR analysis of a proposed, selected actinomycete-specific 439 bp fragment of the 65 kDa heat-shock protein was negative for three of the four isolates. The creation of Segniliparaceae fam. nov. is proposed to encompass the genus Segniliparus gen. nov., including two novel species, the type species Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov., with the respective type strains CDC 1076T (=ATCC BAA-972T=CIP 108378T) and CDC 945T (=ATCC BAA-974T=CIP 108380T).

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