Amplification Of Functional Genes Research Articles

Study of bacterial population dynamics in seed culture developed for ammonia reduction from synthetic wastewater.

The waterbodies have been polluted by various natural and anthropogenic activities. The aquatic waste includes ammonia as one of the most toxic pollutants. Several biological treatment systems involving anoxic and semi anoxic bacteria have been proposed for reducing nitrogen loads from wastewater and increasing the efficiency and cost effectiveness. These bacteria play a vital role in the processes involved in the nitrogen cycle in nature. However, the enrichment, sustainability and identification of bacterial communities for wastewater treatment is an important aspect. Most of the chemolithotrophs are unculturable hence their identification and measurement of abundance remains a challenging task. In this study the different bacteria involved in total nitrogen removal from the wastewater are enriched for 700days under anoxic condition. The synthetic wastewater containing 0.382g/L of ammonium chloride was used. Molecular identification of the bacteria involved in various steps of the nitrogen cycle was carried out based on amplification of functional genes and 16S rRNA gene Polymerase chain reaction followed by DNA sequencing. Change in the abundance of chemolithotrophs was studied using qPCR. The mutual growth of various nitrifiers along with anaerobic bacteria were identified by molecular characterisation of DNA at various time intervals with the different genes involved in the nitrogen cycle. Nitrosomonas species like Nitrosomonas europaea were identified throughout the batch scale studies possessing the genes associated with ammonia oxidizing bacteria and nitrite oxidizing bacteria which act as a complete ammonia oxidizer. The uncultured species of Nitrospira and anammox bacteria were also observed which predicts the coexistence of the anammox and comammox bacteria in a batch scale study. The coexistence of the semi anoxic and anoxic bacteria helped in the growth of these bacteria for a longer duration of time. The nitrite produced by the comammox during nitrification can be utilized by anammox as an electron carrier. The other species of denitrifiers like Pseudomonas denitrificans and Aminobacter aminovorans were also observed. It is concluded that the enrichment of semi anoxic and anoxic bacteria was faster with the increase in growth of the bacteria involved in nitrification, comammox, anammox and partial denitrification process. The bacterial growth is enhanced and the efficiency is increased which can be further used in the development of small pilot scale bioreactor for total nitrogen removal.

Graphitic carbon nitride alleviates cadmium toxicity to microbial communities in soybean rhizosphere.

Cadmium (Cd) contamination has led to various harmful impacts on soil microbial ecosystem, agricultural crops, and thus human health. Nanomaterials are promising candidates for reducing the accumulation of heavy metals in plants. In this study, graphitic carbon nitride (g-C3N4), a two-dimensional polymeric nanomaterial, was applied for ameliorating Cd phytotoxicity to soybean (Glycine max (L.) Merr.). Its impacts on rhizosphere variables, microorganisms, and metabolism were examined. It was found that g-C3N4 increased carbon/nitrogen/phosphorus (C/N/P) content, especially when N contents were averagely 4.2 times higher in the g-C3N4-treated groups. g-C3N4 significantly induced alterations in microbial community structures (P < 0.05). The abundance of the probiotics class Nitrososphaeria was enriched (on average 70% higher in the g-C3N4-treated groups) as was Actinobacteria (226% higher in the g-C3N4 group than in the CK group). At the genus level, g-C3N4 recruited more Bradyrhizobium (122% higher) in the Cd + g-C3N4 group than in the Cd group and more Sphingomonas (on average 24% higher) in the g-C3N4-treated groups. The changes of microbial clusters demonstrated the potential of g-C3N4 to shape microbial functions, promote plant growth, and enhance Cd resistance, despite observing less pronounced modifications in microbial communities in Cd-contaminated soil compared to Cd-free soil. Moreover, abundance of functional genes related to C/N/P transformation was more significantly promoted by g-C3N4 in Cd-contaminated soil (increased by 146%) than in Cd-free one (increased by 32.8%). Therefore, g-C3N4 facilitated enhanced microbial survival and adaptation through the amplification of functional genes. These results validated the alleviation of g-C3N4 on the microbial communities in the soybean rhizosphere and shed a new light on the application of environmental-friendly nanomaterials for secure production of the crop under soil Cd exposure.

A novel Pseudomonas aeruginosa strain performs simultaneous heterotrophic nitrification-aerobic denitrification and aerobic phosphate removal

Nitrogen and phosphate removal from wastewater relies on different functional bacteria. In this study, a novel strain affiliated with Pseudomonas aeruginosa was isolated from activated sludge by gradient dilution and performed heterotrophic nitrification-aerobic denitrification and aerobic phosphate removal (HNADPR). The strain showed an ammonium removal efficiency of 87% and a phosphate removal efficiency of 97% under optimal conditions, such as C/N ratio of 10, P/N ratio of 0.1, temperature of 30°C, and pH of 7.5–8.5. The modified Gompertz model could fit well the heterotrophic ammonium nitrification, aerobic nitrite/nitrate denitrification, and aerobic phosphate removal processes. Functional gene amplification indicated that ammonium removal followed the complete HN-AD pathway (NH4+ → NH2OH → NO2− → NO3− → NO2− → NO → N2O → N2). Phosphate removal only occurred under aerobic conditions and ceased under anaerobic conditions. In successive aerobic cycles, the strain persistently took up phosphate. In wastewater, phosphate was aerobically converted into cell membrane, intracellular and extracellular polymeric substrates (EPS). Phosphorus in the form of phosphate monoester was pooled in EPS. A hypothetic aerobic phosphate removal model for strain SNDPR-01 is proposed to improve our understanding of the novel bacterial function of HNADPR.

Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) pathway dominates nitrate reduction processes in rhizosphere and non-rhizosphere of four fertilized farmland soil

Nitrate (NO3−) reduction partitioning between denitrification, anaerobic ammonium oxidation (anammox), denitrifying anaerobic methane oxidation (DAMO), and dissimilatory nitrate reduction to ammonium (DNRA), can influence the nitrogen (N) use efficiency and crop production in arid farmland. The microbial structure, function and potential rates of denitrification, anammox, DAMO and DNRA, and their respective contributions to total NO3− reduction were investigated in rhizosphere and non-rhizosphere soil of four typical crops in north China by functional gene amplification, high-throughput sequencing, network analysis and isotopic tracing technique. The measured denitrification and DNRA rate varied from 0.0294 to 20.769 nmol N g−1 h−1and 2.4125–58.682 nmol N g−1 h−1, respectively, based on which DNRA pathway contributed to 84.44 ± 14.40% of dissimilatory NO3− reduction, hence dominated NO3− reduction processes compared to denitrification. Anammox and DAMO were not detected. High-throughput sequencing analysis on DNRA nrfA gene, and denitrification nirS and nirK genes demonstrated that these two processes did not correlate to corresponding gene abundance or dominant genus. RDA and Pearson's correlation analysis illustrated that DNRA rate was significantly correlated with the abundance of Chthiniobacter, as well as total organic matter (TOM); denitrification rate was significantly correlated with the abundance of Lautropia, so did TOM. Network analysis showed that the genus performed DNRA was the key connector in the microbial community of dissimilatory nitrate reducers. This study simultaneously investigated the dissimilatory nitrate reduction processes in rhizosphere and non-rhizosphere soils in arid farmland, highlighting that DNRA dominated NO3− reduction processes against denitrification. As denitrification results in N loss, whereas DNRA contributes to N retention, the relative contributions of DNRA versus denitrification activities should be considered appropriately when assessing N transformation processes and N fertilizer management in arid farmland fields.

Partitioning between atmospheric deposition and canopy microbial nitrification into throughfall nitrate fluxes in a Mediterranean forest

Abstract Microbial activity plays a central role in nitrogen (N) cycling, with effects on forest productivity. Although N biotransformations, such as nitrification, are known to occur in the soil, here we investigate whether nitrifiers are present in tree canopies and actively process atmospheric N. This study was conducted in a Mediterranean holm oak (Quercus ilex L.) forest in Spain during the transition from hot dry summer to cool wet winter. We quantified —N and —N fluxes for rainfall (RF) and throughfall (TF) and used δ15N, δ18O and Δ17O to elucidate sources of . Finally, we characterized microbial communities and abundance of nitrifiers on foliage, RF and TF water through metabarcoding and quantitative polymerase chain reaction respectively. NO3—N fluxes at the site were larger in TF than RF, suggesting a contribution from dry deposition, as also supported by δ15N and δ18O. However, Δ17O indicated that about 20% of in TF derived from canopies nitrification in August, after a severe drought, with a lower proportion in September (≈8%). This seasonal partitioning between biologically and atmospherically derived coincided with a decreasing trend of the abundance of archaeal nitrifiers. Tree canopies and TF had more diverse microbial communities than RF. Yet, RF showed higher variability in microbial composition, likely associated with the origin of air masses. Synthesis. Atmospheric N deposition is significantly altered after passing through tree canopies. While nitrification has been proposed as one of the mechanisms responsible for these changes, very few studies directly investigate its occurrence. Here, we showed that nitrification by epiphytic leaf microbes contributed to increasing NO3 in TF and that nitrifiers' activity was reduced going from the dry and hot summer to the cool winter. Overall, these results highlight the power of coupling microbial community analysis, functional gene amplification and stable isotope approaches to examine ecosystem‐scale processes.

Improvements of PCR Amplification of Guanine plus Cytosine-Rich Constructs of Mycobacterium tuberculosis Gene using DMSO

Vaccine research entered a new era when several useful molecular research tools were established. Instead of attenuated virulent microorganisms or killed virulent microorganisms, effective subunit vaccines were developed using recombinant DNA technology. By using the technology, selected genes of the virulent microorganisms can be amplified, cloned, expressed, and evaluated as vaccine components in challenge studies. However, a major bottleneck with the amplification of functional genes from Mycobacterium&#x0D; tubeculosis containing guanine plus cytosin-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting temperatures. To solve this problem in this research, the amplification reaction was modified by addition of dimethyl sulfoxide (DMSO) into amplification reaction mixtures. It was found that 10% (v/v) of DMSO in the reaction mixture improved the amplification of GC-rich template of M. tuberculosis gene. This result indicating that amplification of unbalanced content of G and C deoxyribonucleotides genome could be improved using low-cost organic molecule, DMSO. Therefore, the DMSO should be widely useful as an enhancer to improve the amplification of GC rich construct from other genome.&#x0D; Keywords: Mycobacterium tubeculosis,vaccine, dimethyl sulfoxide, Guanine-Cytosine

Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes

The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes.

Diverse arsenic- and iron-cycling microbial communities in arsenic-contaminated aquifers used for drinking water in Bangladesh.

Subsurface removal of arsenic by injection with oxygenated groundwater has been proposed as a viable technology for obtaining 'safe' drinking water in Bangladesh. While the oxidation of ferrous iron to solid ferric iron minerals, to which arsenic adsorbs, is assumed to be driven by abiotic reactions, metal-cycling microorganisms may potentially affect arsenic removal. A cultivation-independent survey covering 24 drinking water wells in several geographical regions in Bangladesh was conducted to obtain information on microbial community structure and diversity in general, and on specific functional groups capable of the oxidation or reduction of arsenic or iron. Each functional group, targeted by either group-specific 16S rRNA or functional gene amplification, occurred in at least 79% of investigated samples. Putative arsenate reducers and iron-oxidizing Gallionellaceae were present at low diversity, while more variation in potentially arsenite-oxidizing microorganisms and iron-reducing Desulfuromonadales was revealed within and between samples. Relations between community composition on the one hand and hydrochemistry on the other hand were in general not evident, apart from an impact of salinity on iron-cycling microorganisms. Our data suggest widespread potential for a positive contribution of arsenite and iron oxidizers to arsenic removal upon injection with oxygenated water, but also indicate a potential risk for arsenic re-mobilization by anaerobic arsenate and iron reducers once injection is halted.

Ammonia-oxidizing Archaea in Kamchatka Hot Springs

Mounting evidence suggests that ammonia-oxidizing archaea (AOA) may play important roles in nitrogen cycling in geothermal environments. In this study, the diversity, distribution and ecological significance of AOA in terrestrial hot springs in Kamchatka (Far East Russia) were explored using amoA genes complemented by analysis of glycerol dialkyl glycerol tetraethers (GDGTs) of archaea. PCR amplification of functional genes (amoA) from AOA and ammonia-oxidizing bacteria (AOB) was performed on microbial mats/streamers and sediments collected from three hot springs (42°C to 87°C and pH 5.5-7.0). No amoA genes of AOB were detected. The amoA genes of AOA formed three distinct phylogenetic clusters with Cluster 3 representing the majority (∼59%) of OTUs. Some of the sequences from Cluster 3 were closely related to those from acidic soil environments, which is consistent with the predominance of low pH (<7.0) in these hot springs. Species richness (estimated by Chao1) was more frequently higher at temperatures below 75°C than above it, indicating that AOA may be favored in the moderately high temperature environments. Quantitative PCR of 16S rRNA genes showed that crenarchaeota counted for up to 80% of total archaea. S-LIBSHUFF separated all samples into two phylogenetic groups. The profiles of GDGTs were well separated among the studied springs, suggesting a spatial patterning of archaeal lipid biomarkers. However, this patterning did not correlate significantly with variation in archaeal amoA, suggesting that AOA are not the predominant archaeal group in these springs producing the observed GDGTs.

Megakaryocyte polyploidization is associated with a functional gene amplification

It is believed that polyploidy induces an orchestrated increase in gene expression. To know whether all alleles remain functional during megakaryocyte polyploidization, we used a well-established fluorescence in situ hybridization technique which allows one to simultaneously detect pre-mRNAs and assess ploidy level in a single cell. All alleles of GPIIb, GPIIIa, VWF, beta-actin, hsp70, c-mpl, Fli-1, and FOG-1 genes are transcriptionally active in megakaryocytes from 4N to 32N. All X chromosomes in male cells are transcriptionally active but only half of them are transcriptionally active in female megakaryocytes, as revealed by the transcriptional activity of the GATA-1 gene. Nuclear untranslated XIST RNA accumulates on the inactivated X chromosomes, indicating that they are subjected to a normal inactivation process. Altogether, our results demonstrate that megakaryocyte polyploidization results in a functional gene amplification whose likely function is an increase in protein synthesis parallel with cell enlargement.