rRNA Gene Amplification Research Articles

Isolation and selection of spore-forming bacilli with potential for self-healing of concrete

The self-healing of concrete through the use of bacteria is an ecological, sustainable, and environmentally friendly method that can replace time-consuming and costly repairs. The aim of this research was to isolate and select spore-forming bacilli with potential for concrete self-healing. Soil samples from the limestone mines of Simbal, Trujillo, La Libertad, Peru were used. These samples were processed to primarily isolate spore-forming bacteria. The self-healing potential of these bacteria was evaluated through their ability to produce the urease enzyme, grow at an alkaline pH, and produce calcium carbonate crystals in a culture medium with urea and CaCl2 at pH 8.0. The produced crystals were visualized with scanning electron microscopy and their calcium content was confirmed through EDS analysis. The isolated bacteria were identified by 16 S rRNA gene amplification by PCR as Bacillus sp and Streptomyces sp. It was observed that Bacillus sp. produces CaCO3 crystals with vaterite morphology, while Streptomyces sp. produces rhombohedral calcite crystals. It is concluded that the isolated bacteria have the potential to be used in concrete self-healing processes.

Production, purification and characterization of endo-polygalacturonase using novel strain of Bacillus pumilus through RSM

Polygalacturonases (PG) are the enzymes that cause depolymerization of pectin. In current study, bacterial strain was isolated from rotten sample and then purified. It was screened for endo-polygalacturonase activity using PSAM media. It had shown positive response for endo-PG activity. Bacterial DNA was isolated and 16 S rRNA gene amplification was done using universal primer pair of 8 F and 1492 R. Bacterial strain was also amplified by 16 S rRNA gene for sequencing. BLAST of gene sequence on NCBI database had shown that bacterial isolate was identified as Bacillus pumilus. Endo-polygalacturonase enzyme was produced by submerged fermentation to optimize culture conditions by RSM in JMP-12 software. The optimized parameters had shown maximum endo-polygalacturonase activity of 153.2 U/mL/min after using 3 g of orange peels as substrate, 3 mL of inoculum size, 6.5 pH buffer, 40°C temperature and 3 days of incubation. After optimizing fermentation conditions, endo-polygalacturonase was precipitated using 70 % concentration of ammonium sulfate. This partially precipitated sample was dialyzed with dialysis tube and used to find activity of endo-polygalacturonase (1620.6 U/mL/min). This sample was applied to gel filtration chromatography for further purification. Endo-polygalacturonase activity increased many times 2231.44 U/mL/min after gel filtration. The Molecular weight of endo-polygalacturonase was 46 kDa after SDS PAGE characterization. High Vmax value and alkaliphilic nature of endo-polygalacturonase will make it good candidate for food and feed industry near future.

Cecal Microbial Hydrogen Cycling Potential Is Linked to Feed Efficiency Phenotypes in Chickens.

In chickens, early life exposure to environmental microbes has long-lasting impacts on gastrointestinal (GI) microbiome development and host health and growth, via mechanisms that remain uncharacterized. In this study, we demonstrated that administrating a fecal microbiome transplant (FMT) from adults to day-of-hatch chicks results in significantly higher body mass of birds and decreased residual feed intake (RFI), implying enhanced feed efficiency, at 6 weeks of age. To assess the potential mechanisms through which FMT affects adult bird phenotype, we combined 16 S rRNA gene amplification, metagenomic, and comparative genomic approaches to survey the composition and predicted activities of the resident microbiome of various GI tract segments. Early life FMT exposure had a long-lasting significant effect on the microbial community composition and function of the ceca but not on other GI segments. Within the ceca of 6-week-old FMT birds, hydrogenotrophic microbial lineages and genes were most differentially enriched. The results suggest that thermodynamic regulation in the cecum, in this case via hydrogenotrophic methanogenic and sulfur-cycling lineages, potentially serving as hydrogen sinks, may enhance fermentative efficiency and dietary energy harvest capacity. Our study provides a specific mechanism of action through which early-life microbiome transplants modulate market-relevant phenotypes in poultry and, thereby, may represent a significant advance toward microbiome-focused sustainable agriculture.

Chronic stress and captivity alter the cloacal microbiome of a wild songbird

There are complex interactions between an organism's microbiome and its response to stressors, often referred to as the 'gut-brain axis'; however, the ecological relevance of this axis in wild animals remains poorly understood. Here, we used a chronic mild stress protocol to induce stress in wild-caught house sparrows (Passer domesticus), and compared microbial communities among stressed animals, those recovering from stress, captive controls (unstressed) and a group not brought into captivity. We assessed changes in microbial communities and abundance of shed microbes by culturing cloacal samples on multiple media to select for aerobic and anaerobic bacteria and fungi. We complemented this with cultivation-independent 16S and ITS rRNA gene amplification and sequencing, pairing these results with host physiological and immune metrics, including body mass change, relative spleen mass and plasma corticosterone concentrations. We found significant effects of stress and captivity on the house sparrow microbiomes, with stress leading to an increased relative abundance of endotoxin-producing bacteria - a possible mechanism for the hyperinflammatory response observed in captive avians. While we found evidence that the microbiome community partially recovers after stress cessation, animals may lose key taxa, and the abundance of endotoxin-producing bacteria persists. Our results suggest an overall link between chronic stress, host immune system and the microbiome, with the loss of potentially beneficial taxa (e.g. lactic acid bacteria), and an increase in endotoxin-producing bacteria due to stress and captivity. Ultimately, consideration of the host's microbiome may be useful when evaluating the impact of stressors on individual and population health.

Microbiota of a UASB Reactor Treating Palm Oil Mill Effluent Using HiSeq Sequencing

Potential microbial communities in the UASB reactor fed with two different substrates i.e synthetic wastewater and raw palm oil mill effluent (POME) were elucidated by using one of the most popular techniques in molecular methods, viz 16S rDNA cloning. The methodology involved; the extraction of nucleic acids, amplification and cloning of the 16S rRNA genes on sequencing HiSeq platform and finally identification and affiliation of the isolated clone with the aid of phylogenetic software. Results showed that the genus methanosarcina and methanosaeta were dominant methanogens in this study for both substrates types. Overall, microbial population (Bacteria and Archaea) in sample A (POME as substrate) is more diverse compared to sample B (synthetic wastewater as substrate) due to abundance of microorganism population in raw POME which was used as a substrate. However for the methanogenic (Archaea) diversity in both samples, there was not much different between sample A and sample B probably due to similar inoculum was inoculated in the reactor despite of have using different substrate type.

Validation of bacterial markers to discriminate against the source of nitrate contamination: a promising application within the EU Nitrates Directive

The European Community (EC) Nitrates Directive (ND) (Council Directive 1991/676/EEC) aims to avoid water pollution by nitrates from poor farming practices. Under the ND, Nitrate Vulnerable Zones (NVZs) have been defined as areas where the concentration of agricultural nitrates in surface or groundwater exceeds 50 mg/L. Groundwater is one of the main sources of drinking water in Europe, so ensuring its quality is of significant importance. Monitoring of water parameters, the identification of pollution, the development of good agricultural practice codes is included in ND as measures and action programs. ND provides for the attribution of areas affected by nitrate pollution to the NVZ in which farmers must comply with measures necessary to reduce nitrogen in excess and protect the quality of environmental matrices. However, in some cases the quality of water bodies remains poor, suggesting that some action measures are ineffective or need improvements. Nevertheless, nitrates in excess in groundwater might be related to industrial inputs, untreated waste discharge, and sewage spilling. Due to this, identifying non-agricultural contributions to nitrate groundwater pollution can be of great interest. With this aim, the employment of biomarkers such as microbes associated with the intestinal tract of a specific host is under study for identifying fecal pollution. This strategy is known, in scientific literature, as Microbial Source Tracking (MST)(Furtula et al. 2011)The amplification of 16S rRNA genes of host-specific Bacteroidales allows discriminating against human and livestock fecal sources in samples from nitrate polluted environment. The aim of this work is to evaluate the sensitivity and specificity of these intestinal biomarkers in groundwater samples collected along with the Apulian Region (Southern Italy) in order to assess the applicability of the MST in the employ of ND. A preliminary study was carried out to validate the performance of seven markers for MST using two different approaches: Polimerase Chain Reaction (PCR) and quantitative Real-time PCR (qPCR) assays. For both PCR and qPCR experiments selected primer sets were checked using fecal samples of known origin as positive controls. PCR assay was used for human (HF74 and Enterococcus faecalis) (Bernhard and Field 2000, Jackson et al. 2004), cattle (CF123) (Bernhard and Field 2000), equine (HoF597) (Dick et al. 2005), and pig (Pig163) (Dick et al. 2005) markers screening. Two additional markers were tested through Real-time PCR: human (Human- Bacteroides) (Seurinck et al. 2005) and zootechnical (BacPre I) (Kobayashi et al. 2012). The results indicate that biomarkers can be considered reliable in distinguishing human from animal pollution. Nevertheless, our studies show that the tests conducted with the human Enterococcus faecalis biomarker do not discriminate the zootechnical source from the human one. In some cases, PCR-tested biomarkers cannot determine the source of contamination in environmental matrices due to the detection limit. Animal and human fecal markers were widely detected on eleven groundwater samples through Real Time PCR, highlighting the prevalent source of contamination in the environmental matrix. This research provides evidence that MST technology is a valid tool for local authorities to identify the source of nitrate contamination and review uncertainties during the NVZs definition and the action program development required by the European Nitrate Directive.

Biosynthesis of TiO2 nanoparticles using prodigiosin and evaluating its antibacterial activity against biofilm producing MDR- Acinetobacter baumannii

A rising number of hospital infections were caused by multi drug resistant A.baumannii. This microorganism has become a big global concern for clinicians. This study aimed to evaluate the antimicrobial activity of biosynthesized TiO2 nanoparticles against biofilm producing multi drug resistant A. baumannii. Bacteria were isolated from burn wounds. The selected isolate was identified using the routine biochemical assays, viteck 2, and confirmed by PCR technique, targeting the 16S rRNA and blaOXA-51 genes. Antimicrobial susceptibility tests were performed using Viteck 2 system and the biofilm production was tested by using microtiter plate method. S marcescens was used for production of the prodigiosin which characterized later by UV-visible spectroscopy and then was used for biosynthesis of titanium dioxide nanoparticles (TiO2) NPs. Atomic force microscopy, X-ray diffractometer and field emission scanning electron microscopy were used for characterization of TiO2 NPs. Antimicrobial activity of TiO2 NPs was examined by well diffusion assay using concentration of 0.4- 0.006 mg/ml. The studied isolate was beta-lactamase producer and showed resistance to aminoglycosides, quinolones, furanes and trimethoprim/ sulphonamide, PCR amplification of 16S rRNA and blaOXA-51 genes was used for detection of A baumannii. The selected isolate was a strong biofilm producer with 5.9 times more than the OD values of the control. Atomic force microscopy images showed that the synthesized TiO2 NPs were in spherical shape with an average diameter of 67.49 nm. The TiO2 NPs inhibited the bacterial growth at concentrations of ≥ 0.1mg/ ml and a maximum zone of inhibition recorded was 22 mm at concentration of 0.4 mg/ ml. Biosynthesis of TiO2 NPs using prodigiosin was showed a promising antibacterial activity against strong biofilm producing MDR- A. baumannii.

Rhino-orbital mucormycosis due to Apophysomyces ossiformis in a patient with diabetes mellitus: a case report

BackgroundThe most common aetiological agents of mucormycosis are Rhizopus, Mucor, Apophysomyces and Lichtheimia. Apophysomyces is comparatively rare, as it has been reported in less than 3% of mucormycosis cases. The genus Apophysomyces includes six species, and only A. elegans, A. mexicanus, A. variabilis and A. ossiformis have been reported to cause infections in both immunocompetent and immunocompromised patients.Case presentationWe present a case of a 46-year-old male patient with bilateral blepharoedema, corneal opacity in the left eye and poorly controlled diabetes mellitus. The patient was subjected to total maxillectomy, exenteration of the left orbit and treatment with liposomal amphotericin B. Direct mycological analysis with KOH 10% revealed hyaline, coenocytic, long and wide hyphae. Apophysomyces ossiformis was identified from maxillary biopsy using 18S-ITS1–5.8S-ITS2-28S rRNA gene amplification and sequencing. The patient requested to be transferred to another hospital to continue treatment, where he died on the ninth day after admittance.ConclusionTo the best of our knowledge, this is the first case of rhino-orbital mucormycosis due to A. ossiformis with a fatal outcome. This case reveals the need to identify the fungus causing mucormycosis with molecular methods to identify adequate treatment therapies for patients with this infection.

DETECTION OF CAMPYLOBACTER SPP. WITH REAL-TIME POLYMERASE CHAIN REACTION

Bacteria of Campylobacter genus are ones of the main zoonotic pathogens causing human and animal diseases. Campylobacter organisms are microaerophiles and, therefore, require low oxygen concentration (3–5%) and high carbon dioxide concentration (3–10%) for their growth. They use amino acids rather than carbons as a source of energy. Classical bacteriological methods for Campylobacter spp. detection are not always successful due to diffi culties in creating optimal conditions for their growth. Therewith, development and implementation of molecular methods for Campylobacter detection and identifi cation are of current importance. Assay for qualitative Campylobacter spp. detection with real-time polymerase chain reaction using CFX-96 thermocycler was optimized. Highly specifi c segment of 16S rRNA gene allowing identifi cation of 6 Campylobacter species: C. jejuni, C. coli, C. lari, C. upsaliensis, C. helveticus и C. hyointestinalis was selected as an amplifi cation target. Optimal magnesium ion concentration (2.5 мМ) and primer annealing temperature (58 °С) were determined. Eighteen reference strains of various bacteria were tested. Only tests of Campylobacter genus strains gave positive results. The method sensitivity was 40 target molecules. The said method was used for testing 76 samples of raw materials of animal origin. Campylobacter spp. genome was detected in 18 samples. Obtained results showed that the optimized variant of real-time polymerase chain reaction based on 16S rRNA gene amplifi cation was a specifi c, sensitive, rapid, reproducible and accurate method for qualitative detection of Campylobacter spp. in samples of raw animal materials.

Comparative 16S rDNA metagenomics study of two samples of cassava peel heap from Nigeria and India.

The microbiology of many cassava products and the wastes generated during the processing have been reported; however, majority of these reports used culture-dependent methods. This has resulted in a dearth of information on the bacterial diversity of cassava peels and peel heaps. Large amounts of cassava peels generated during the processing of cassava root are usually discharged on land or water as wastes and are allowed to rot in the open, especially in some developing countries. Culture-independent methods such as PCR-based amplification and sequencing of 16S rRNA genes, among others have been used in recent times to study the diversity of microbes in different environmental samples. In this study, bacterial isolates were screened for cellulase and xylanase enzyme activities on minimal agar and genomic DNA was isolated from cassava peel samples; metagenomics was carried out using MiSeq2 × 300 with primers specific for V3-V4 bacterial region. Samples collected from Nigeria (AAG) had more species compared with samples from India (JHA) with 793 and 525 observed OTUs (operational taxonomic units), respectively. Five bacterial isolates from cassava peel-heap samples obtained from Ogbomoso, Nigeria showed no ability to produce cellulase enzyme, seven isolates from the Nigeria samples and three from Jorhat samples were positive for xylanase production; the highest amylase activity was shown by isolate AG18 (10,055U/mL), while the lowest was recorded for isolate JA2 (2333U/mL) with a significant difference observed in the amylase activities of isolates (p ≤ 0.05). Comparing the most abundant taxonomy for each of the samples at different taxonomic levels, the most abundant for sample AAG were phylum Firmicutes (42.11%), class Bacilli (41.27%), order Lactobacillales (33.11%), family Acetobacteraceae (31.30%), genus Acetobacter (30.02%) and unclassified species of Acetobacter (29.88%), while sample JHA had Actinobacteria (47.47%) as the highest phylum and class, order Actinomycetales (47.47%), family Brevibacteriaceae (46.97%), genus Brevibacterium (46.97%) and unclassified species of Brevibacterium (46.89%). This study provides an insight into the vast diversity of the bacteria associated with cassava peel heaps and the ability of some of the bacteria to produce selected extracellular enzymes.

Diversity of Thermophilic Iron-Pyrite-Oxidizing Enrichments from Solfataric Hot Springs in the Chilean Altiplano

Relatively little is known about the microbial communities present in natural environments that meet physico-chemical conditions for the development of potential leaching microorganisms such as thermal ecosystems from the Chilean Altiplano. Thermophilic leaching enrichments were obtained and identified from a high altitude solfataric pound in Lirima hot springs in the Chilean Altiplano. This ecosystem is characterized by hot underground freshwaters, enriched in sulfur compounds showing pH from neutral to acidic. Microbial diversity has been scarcely explored here, and preliminary results demonstrate that hydrothermal pounds are represented by thermophilic anaerobic and acidophilic taxa. Thermophilic leaching cultures in shake flasks were obtained using ferrous iron and pyrite as energy source. The presence of Bacteria and Archaea in oxidizing enrichments was determined by PCR amplification of 16S rRNA genes. A preliminary analysis of microbial diversity using massive sequencing revealed that Bacteria were more abundant than Archaea in both enrichments. Specifically for the iron-oxidizing culture, the majority of the sequences clustered within the Proteobacteria phylum (79%). Among Proteobacteria, the proportion of Betaproteobacteria (42.2%) and Gammaproteobacteria (21.27%) was much higher than that of Alphaproteobacteria (15.5%). Within the Betaproteobacteria class, the most frequent genus was Leptothrix-like. Similar results were obtained for the pyrite oxidizing culture. Interestingly, this study shows the presence of microorganisms close to the Leptothrix genus under low pH conditions (1.7-2.8) and their capacity to grow at high temperatures with ferrous iron or pyrite as sole energy source

Methodological flaws introduce strong bias into molecular analysis of microbial populations

In this study, we report how different cell disruption methods, PCR primers and in silico analyses can seriously bias results from microbial population studies, with consequences for the credibility and reproducibility of the findings. Our results emphasize the pitfalls of commonly used experimental methods that can seriously weaken the interpretation of results. Four different cell lysis methods, three commonly used primer pairs and various computer-based analyses were applied to investigate the microbial diversity of a fermentation sample composed of chicken dung. The fault-prone, but still frequently used, amplified rRNA gene restriction analysis was chosen to identify common weaknesses. In contrast to other studies, we focused on the complete analytical process, from cell disruption to in silico analysis, and identified potential error rates. This identified a wide disagreement of results between applied experimental approaches leading to very different community structures depending on the chosen approach. The interpretation of microbial diversity data remains a challenge. In order to accurately investigate the taxonomic diversity and structure of prokaryotic communities, we suggest a multi-level approach combining DNA-based and DNA-independent techniques. The identified weaknesses of commonly used methods to study microbial diversity can be overcome by a multi-level approach, which produces more reliable data about the fate and behaviour of microbial communities of engineered habitats such as biogas plants, so that the best performance can be ensured.

Cultivation of Anaplasma ovis in the HL-60 human promyelocytic leukemia cell line

The tick-borne bacterium Anaplasma ovis is a widely distributed pathogen affecting sheep, goats and wild ruminants. Here, the HL-60 human promyelocytic leukemia cell line was used to isolate A. ovis from PCR-positive sheep and goats in Heilongjiang Province, China. Two weeks after inoculation, morulae were observed in cytoplasmic vacuoles in four different HL-60 cultures. Confocal microscopy using a Cy3-labeled A. ovis-specific probe confirmed that the HL-60 cells were infected with A. ovis. Cells from the 6th HL-60 subculture displayed positive fluorescence when incubated with A. ovis antiserum in the indirect fluorescent antibody assay. PCR amplification and sequencing of 16S rRNA, groEL, gltA, msp2 and msp4 Anaplasma genes revealed that the four A. ovis culture isolates were identical. Phylogenetic analysis showed that the sequences clustered with other A. ovis strains but could clearly be distinguished from other Anaplasma species. When the 18th subculture of infected HL-60 cells was examined by electron microscopy, lysosomes were often observed near the vacuoles. After the 24th subculture, Giemsa staining and PCR indicated that the HL-60 cells were negative for A. ovis. Although A. ovis can infect HL-60 cells for only four months, the ability of the organism to infect and multiply in HL-60 cells provides a tool to study intra-erythrocytic Anaplasma and host cell interactions.Emerging Microbes & Infections (2017) 6, e83; doi:10.1038/emi.2017.70; published online 20 September 2017

Antimicrobial Potential, Identification and Phylogenetic Affiliation of Wild Mushrooms from Two Sub-Tropical Semi-Evergreen Indian Forest Ecosystems.

The diversity of wild mushrooms was investigated from two protected forest areas in India and 231 mushroom specimens were morphologically identified. Among them, 76 isolates were screened for their antimicrobial potential against seven bacterial and fungal pathogens. Out of 76 isolates, 45 isolates which displayed significant antimicrobial activities were identified using ITS rRNA gene amplification and subsequently phylogenetically characterized using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Sequencing of the ITS rRNA region classified the isolates into 16 genera belonging to 11 families. In total, 11 RAPD and 10 ISSR primers were selected to evaluate genetic diversity based on their banding profile produced. In total 337 RAPD and 312 ISSR bands were detected, among which percentage of polymorphism ranges from 34.2% to 78.8% and 38.6% to 92.4% by using RAPD and ISSR primers respectively. Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) trees of selected two methods were structured similarly, grouping the 46 isolates into two clusters which clearly showed a significant genetic distance among the different strains of wild mushroom, with an similarity coefficient ranges from 0.58 to 1.00 and 0.59 to 1.00 with RAPD and ISSR analysis respectively. This reporthas highlighted both DTR and MNP forests provide a habitat for diverse macrofungal species, therefore having the potential to be used for the discovery of antimicrobials. The report has also demonstrated that both RAPD and ISSR could efficiently differentiate wild mushrooms and could thus be considered as efficient markers for surveying genetic diversity. Additionally, selected six wild edible mushroom strains (Schizophyllum commune BPSM01, Panusgiganteus BPSM27, Pleurotussp. BPSM34, Lentinussp. BPSM37, Pleurotusdjamor BPSM41 and Lentinula sp. BPSM45) were analysed for their nutritional (proteins, carbohydrates, fat and ash content), antioxidant potential. The present findings also suggested that the wild edible mushroom strains do not have only nutritional values but also can be used as an accessible source of natural antioxidants.

Effects of bitter melon (Momordica charantia L.) on the gut microbiota in high fat diet and low dose streptozocin-induced rats

The effects on gut microbiota of type 2 diabetic rats fed a bitter melon formulation (BLSP, a lyophilized superfine powder) were investigated. BLSP treatment significantly reduced fasting blood glucose levels (p < 0.05) and serum insulin levels (p < 0.05) of the diabetic rats. The gut microbiota of treated and control rats were profiled by PCR amplification and pyrosequencing of 16S rRNA genes (V3–V9 region). BLSP significantly reduced the ratio of Firmicutes to Bacteroidetes in diabetic rats, while the relative abundances of Ruminococcaceae, Bacteroides and Ruminococcus were significantly lowered in BLSP-treated rats compared to diabetic rats. Additionally, BLSP significantly suppressed the activation of MAPK (JNK and p38). The results indicate that BLSP can significantly modify the proportions of particular gut microbiota in diabetic rats without disturbing the normal population diversity. By suppressing the activation of MAPK signaling pathway, a BLSP containing diet may ameliorate type 2 diabetes.

PCR method for the rapid detection and discrimination of Legionella spp. based on the amplification of pcs, pmtA, and 16S rRNA genes.

Legionella bacteria are organisms of public health interest due to their ability to cause pneumonia (Legionnaires' disease) in susceptible humans and their ubiquitous presence in water supply systems. Rapid diagnosis of Legionnaires' disease allows the use of therapy specific for the disease. L. pneumophila serogroup 1 is the most common cause of infection acquired in community and hospital environments. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this work, simplex and duplex PCR assays with the use of new molecular markers pcs and pmtA involved in phosphatidylcholine synthesis were specified for rapid and cost-efficient identification and distinguishing Legionella species. The sets of primers developed were found to be sensitive and specific for reliable detection of Legionella belonging to the eight most clinically relevant species. Among these, four primer sets I, II, VI, and VII used for duplex-PCRs proved to have the highest identification power and reliability in the detection of the bacteria. Application of this PCR-based method should improve detection of Legionella spp. in both clinical and environmental settings and facilitate molecular typing of these organisms.

Weissella jogaejeotgali sp. nov., isolated from jogae jeotgal, a traditional Korean fermented seafood.

Strain FOL01T was isolated from traditionally fermented Korean jogae jeotgal (fermented clams). Phylogenetic sequence analysis of the 16S rRNA gene from FOL01T revealed that it is closely related to Weissella thailandensis FS61-1T and Weissella paramesenteroides ATCC 33313T with 99.39 % and 98.50 % 16S rRNA gene sequence similarities, respectively. API and VITEK analyses showed that strain FOL01T could be separated from its nearest phylogenetic relatives with respect to carbohydrate fermentation and antibiotic resistance. Subsequent amplified rRNA gene restriction analysis of 16S rRNA genes and HaeIII-restriction enzyme profiling of genomic DNAs revealed different band patterns. In addition, DNA-DNA hybridization of genomic DNAs showed 63.9 % relatedness. Analysis of the composition of cellular fatty acids confirmed that strain FOL01T differs from its close relatives and supports the proposal to assign this organism to a novel species of the genus Weissella. Based on these results, strain FOL01T could be classified as a novel species of the genus Weissella, for which the name Weissella jogaejeotgali sp. nov. is proposed. The type strain is FOL01T ( = KCCM 43128T = JCM 30589T).

Inosine at Different Primer Positions to Study Structure and Diversity of Prokaryotic Populations

Culture-independent methods, employed to study the diversity and complexity of microbial communities that are based on amplification of rRNA genes with universal primers, include gradient gel electrophoresis (denaturing or temperature), single-strand-conformation polymorphism, restriction fragment length polymorphism, qPCR and high-throughput DNA sequencing. Substituting one or more base(s) within or at the 3'-termi of the universal primers by inosine can overcome some of their shortcomings improving amplification capacity. Universal primer sets do not usually amplify sequences with nucleotide mismatch to the templates, particularly in the last three bases, whereas inosine-modified primers anneal and amplify a variety of rRNA gene sequences. Inosine-containing primers are therefore might be useful to detect more species in diverse prokaryotic populations. The article summarizes the pros and cons of using inosine especially at the 3' termini of universal primers in nucleic acid amplification for the study of microbial diversity.

Detection of characteristic heterochromatin distribution, highly amplified rRNA genes and presence of the human satellite III DNA motif in the scleractinian coral Echinophyllia aspera Ellis and Solander 1788

Detection of characteristic heterochromatin distribution, highly amplified rRNA genes and presence of the human satellite III DNA motif in the scleractinian coral Echinophyllia aspera Ellis and Solander 1788

Microbial community structure and nitrogenase gene diversity of sediment from a deep-sea hydrothermal vent field on the Southwest Indian Ridge

A sediment sample was collected from a deep-sea hydrothermal vent field located at a depth of 2 951 m on the Southwest Indian Ridge. Phylogenetic analyses were performed on the prokaryotic community using polymerase chain reaction (PCR) amplification of the 16S rRNA and nifH genes. Within the Archaea, the dominant clones were from marine benthic group E (MBGE) and marine group I (MGI) belonging to the phyla Euryarchaeota and Thaumarchaeota, respectively. More than half of the bacterial clones belonged to the Proteobacteria, and most fell within the Gammaproteobacteria. No epsilonproteobacterial sequence was observed. Additional phyla were detected including the Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Nitrospirae, Chloroflexi, Chlorobi, Chlamydiae, Verrucomicrobia, and candidate divisions OD1, OP11, WS3 and TM6, confirming their existence in hydrothermal vent environments. The detection of nifH gene suggests that biological nitrogen fixation may occur in the hydrothermal vent field of the Southwest Indian Ridge. Phylogenetic analysis indicated that only Clusters I and III NifH were present. This is consistent with the phylogenetic analysis of the microbial 16S rRNA genes, indicating that Bacteria play the main role in nitrogen fixation in this hydrothermal vent environment.