Clone Library Construction Research Articles

Insights into the abundance, expression and diversity of key denitrification genes in an ecologically managed greenhouse agricultural soil

Understanding the bacteria associated with nitrification and denitrification is crucial for comprehending the processes that lead to nitrous oxide emissions in agricultural greenhouse soils. Therefore, it is important to determine their abundance and expression to gain insight into these processes.The aim of this study was to explore the bacterial communities associated with denitrification in a greenhouse agricultural soil amended with crop residues and manure for six years. For this purpose, we proceeded to detect and quantify the genes nirK and nirS and the gene nosZ through clone library construction, sequencing, phylogenetic analysis, and quantitative polymerase chain reaction (qPCR). Sequence analysis based on the clone library revealed that many of the nirS or nirK genes detected were not closely related to known denitrifier bacteria, but some of the nosZ sequences were related to the genera such as Pseudomonas, Halomonas, and Marinobacter. Furthermore, the qPCR revealed a high abundance of DNA copies in nirK, 6.08 × 109 ± 1.16 × 109, while nirS and nosZ showed lower values, 9.05 × 106 ± 1.65 × 106 and 8.71 × 106 ± 1.44 × 106, respectively. However, the highest expression rate was observed for nirS (mRNA/DNA ratio = 3.10 × 10− 3), while nirK and nosZ showed 10-fold lower expression rates (4.4 × 10− 4 and 3.5 × 10− 4, respectively). The results of this work provide a preliminary overview of the diversity, abundance and expression of key genes associated with the denitrification process in this type of soil and are a starting point for further studies to understand how this type of soil management can influence the denitrification process.

Ratio of Histamine-Producing/Non-Histamine-Producing Subgroups of Tetragenococcus halophilus Determines the Histamine Accumulation during Spontaneous Fermentation of Soy Sauce.

Strain specificity (within-species variation) of microorganisms occurs widely in nature. It might affect microbiome construction and function in a complex microbial environment. Tetragenococcus halophilus, a halophilic bacterium that generally is used in high salt food fermentation, consists of two histamine-producing and non-histamine-producing subgroups. It is unclear whether and how the strain specificity of histamine-producing capacity influences the microbial community function during food fermentation. Here, based on systematic bioinformatic analysis, histamine production dynamic analysis, clone library construction analysis, and cultivation-based identification, we identified that T. halophilus is the focal histamine-producing microorganism during soy sauce fermentation. Furthermore, we discovered that a larger number and ratio of histamine-producing subgroups of T. halophilus significantly contributed more histamine production. We were able to artificially decrease the ratio of histamine-producing to non-histamine-producing subgroups of T. halophilus in complex soy sauce microbiota and realized the reduction of histamine by 34%. This study emphasizes the significance of strain specificity in regulating microbiome function. This study investigated how strain specificity influenced microbial community function and developed an efficient technique for histamine control. IMPORTANCE Inhibiting the production of microbiological hazards under the assumption of stable and high-quality fermentation is a critical and time-consuming task for the food fermentation industry. For spontaneously fermented food, it can be realized theoretically by finding and controlling the focal hazard-producing microorganism in complex microbiota. This work used histamine control in soy sauce as a model and developed a system-level approach to identify and regulate the focal hazard-producing microorganism. We discovered that the strain specificity of focal hazard-producing microorganisms had an important impact on hazard accumulation. Microorganisms frequently exhibit strain specificity. Strain specificity is receiving increasing interest since it determines not only microbial robustness but also microbial community assembly and microbiome function. This study creatively explored how the strain specificity of microorganisms influenced microbiome function. In addition, we believe that this work provides an excellent model for microbiological hazard control which can promote future work in other systems.

Hydrology driven vertical distribution of prokaryotes and methane functional groups in a subtropical peatland

Microbiomes play fundamental roles in ecological function of peatlands. Despite the confirmed role of hydrology in the modulation of microbial communities in peatlands, its impact on microbial functional groups involved in methane cycle has remained poorly defined particularly at subtropical peatlands. To this end, prokaryotic communities and methane related functional groups from two peat profiles were investigated in the Dajiuhu Peatland, a subtropical peatland in central China; via high throughput sequencing of the 16S rRNA genes, quantitative PCR, and clone library construction for functional genes for methanogens (mcrA) and methanotrophs (pmoA). Results showed that depth to water table (DWT), indicator for water table variation, significantly impacted the vertical distribution of prokaryotic communities, especially Acidobacteria and Bathyarchaeota, and methane functional groups. FenC, RC I and RRC I dominated methanogens in middle and bottom samples of the profiles, whereas Methylocystis dominated methanotrophic communities in surface and middle samples. Quantitatively methanogens decreased with increasing DWT, whereas reverse pattern was seen for methanotrophs. Aerobic or anaerobic OTUs clustered into different modules in the co-occurrence network, indicating a strongly oxygen-dependent niche preference. Collectively, our observations demonstrated important ecological regulation of DWT on prokaryotes and CH4 related microbiomes in subtropical peatlands, which expanding our understanding of microbial responses and CH4 cycles in the context of environmental changes globally.

Niche Differentiation of Phenol-Degrading Microorganisms in UASB Granular Sludge as Revealed by Fluorescence in situ Hybridization

A microbial community structure of granules harvested from an anaerobic sludge blanket reactor treating phenolic wastewater was investigated using fluorescence in situ hybridization (FISH) and clone library construction. Clones of Syntrophorhabdaceae and Cryptanaerobacter were observed to be responsible for phenol degradation. For accurate taxonomic assignment of Cryptanaerobacter clones, phylogenetic analysis using nearly full-length 16S ribosomal RNA (rRNA) gene sequences was necessary. Three oligonucleotide probes were designed to detect the following three taxonomic groups: Syntrophorhabdaceae, Cryptanaerobacter , and Syntrophus . FISH analysis of thin sections of anaerobic granules showed a random distribution of bacteria and archaea. However, a well-defined distribution of Syntrophorhabdaceae, Cryptanaerobacter , and Syntrophus was observed. Cryptanaerobacter and Syntrophus were found on the outer layer of the granules and were closely associated with each other, while Syntrophorhabdaceae was located in the deeper part of the granules. Such specific distribution of the bacteria is most likely due to their metabolic association and affinity for the substrate. Phenol degradation in the granular sludge was observed to be carried out in the following way. First, Cryptanaerobacter converts phenol to benzoate, which is then degraded by Syntrophus into acetate. This syntrophic degradation of phenol occurs near the surface of the granule, where the phenol concentration is high. In the deeper part of the granule, where the phenol concentration is lower, Syntrophorhabdaceae degrades phenol into acetate. We observed that Syntrophorhabdaceae is less likely to produce benzoate as an intermediate to feed the neighboring organisms, which contradicts the theories presented by previous studies.

New Arsenite Oxidase Gene (aioA) PCR Primers for Assessing Arsenite-Oxidizer Diversity in the Environment Using High-Throughput Sequencing.

Gene encoding the large subunit of As(III) oxidase (AioA), an important component of the microbial As(III) oxidation system, is a widely used biomarker to characterize As(III)-oxidizing communities in the environment. However, many studies were restricted to a few sequences generated by clone libraries and Sanger sequencing, which may have underestimated the diversity of As(III)-oxidizers in natural environments. In this study, we designed a primer pair, 1109F (5′-ATC TGG GGB AAY RAC AAY TA−3′) and 1548R (5′-TTC ATB GAS GTS AGR TTC AT−3′), targeting gene sequence encoding for the conserved molybdopterin center of the AioA protein, yielding amplicons approximately 450 bp in size that are feasible for highly parallel amplicon sequencing. By utilizing in silico analyses and the experimental construction of clone libraries using Sanger sequencing, the specificity and resolution of 1109F/1548R are approximated with two other previously published and commonly used primers, i.e., M1-2F/M3-2R and deg1F/deg1R. With the use of the 1109F/1548R primer pair, the taxonomic composition of the aioA genes was similar both according to the Sanger and next-generation sequencing (NGS) platforms. Furthermore, high-throughput amplicon sequencing using the primer pair, 1109F/1548R, successfully identified the well-known As(III)-oxidizers in paddy soils and sediments, and they also revealed the differences in the community structure and composition of As(III)-oxidizers in above two biotopes. The random forest analysis showed that the dissolved As(III) had the highest relative influence on the Chao1 index of the aioA genes. These observations demonstrate that the newly designed PCR primers enhanced the ability to detect the diversity of aioA-encoding microorganisms in environments using highly parallel short amplicon sequencing.

Cyanobacteria in microbialites of Alchichica Crater Lake: a polyphasic characterization

ABSTRACT We analysed and characterized the cyanobacteria in microbialites of Alchichica crater lake over a depth gradient, from 3 to 30 m. A polyphasic approach was followed using morphometry, optical and epifluorescence microscopy, and molecular methods. Regions V3–V4 of the 16S rRNA conserved for cyanobacteria were amplified and used for clone library construction. Independently, a NGS library was constructed using the V4 region of the 16S rRNA. To complement the analysis, eight monocultures of cyanobacteria were isolated from microbialites, which were also characterized by microscopy and 16S rRNA sequencing. In total, we described 18 genera of cyanobacteria isolated from microbialites of Alchichica crater lake. The distribution pattern suggests that on the surface there were mostly Nostocales and filamentous Synechococcales, while at depth we found single-celled Synechococcales. Pleurocapsales and Chroococcales were found at all depths, increasing in abundance at 20 and 30 m.

Abundance and community structure of ammonium monooxygenase (amoA) genes in the wet season of liaohe estuary sediments

Ammonia oxidation is an important part of the global nitrogen cycle. Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) are important players in the ammoxidation process. In this study, 13 sediment samples from the Liaohe estuary were collected in the wet season. The abundances of 16S rRNA and amoA genes were detected by real-time fluorescence quantitative PCR. The diversity and community structure of AOA and AOB were investigated by constructing clone libraries. The AOA and AOB amoA gene abundances ranged from 3.10 × 106–2.85 × 107 and 6.59 × 105–1.20 × 107 copies/g wet deposit, respectively. At all sites, AOA abundance was greater than AOB (AOA: AOB = 2.2–86.5). The ratio of AOA: archaeal 16S rRNA gene abundances and AOB: bacterial 16S rRNA gene abundances were 4.6 × 10−3–1.5 × 10−2 and 1.5 × 10−6–1.0 × 10−4. Phylogenetic analysis showed that the AOA amoA sequences were mainly Nitrosophaera and Nitrosopumilus, and most of the AOB amoA sequences belong to unclassified bacteria, which mainly were detected in estuarine sediments and wetland soils. AOA and AOB community had an obvious spatial heterogeneity. In addition, canonical correspondence analysis showed that the salinity, ammonia, total phosphorus, total organic carbon, sand, and silt content were the main driven factors of AOA and AOB community structure, in which the sand and silt content had a significant contribution.

The Role of Agriculture in the Dissemination of Class 1 Integrons, Antimicrobial Resistance, and Diversity of Their Gene Cassettes in Southern China.

Integrons are hot spots for acquiring gene cassettes from the environment and play a major role in the bacterial evolution and dissemination of antimicrobial resistance (AMR), thus posing a serious threat. There are currently studies on integrons and antibiotic resistance genes; however, the presence and association of integrons in different agricultural crops and their subsequent dissemination and role in AMR have not been reported previously. This study examines the abundance of integrons, their gene cassette diversity in various crop soils, and their role in the dissemination of AMR in the southern region of China. Samples from different agri-crop soil, such as rice (R.S), sugarcane (S.S), citrus (C.S), banana (B.S), agricultural runoff (the point where the runoff of all sites meet (R.O)), and wild (non-agricultural) soil (W.S), were collected. Quantitative PCR was used to determine the abundance of integrons, and clone libraries were constructed to examine the gene cassette arrays. All the tested samples were found positive for Class-I (CL1) integrons and revealed a higher concentration and higher relative abundance of R.S than the others, with the least found at the W.S site. The W.S CL1 cassette arrays were found empty, and no putative conserved domains were found. The R.O was found to contain a high number of gene cassettes with various functions, while the smallest number of gene cassettes was found in the S.S among the crop soils. Most of the gene cassettes presented by the R.O were primarily shared with other sites, and the antibiotic-resistant genes were consistently observed to be dominant. The constructed clone libraries represented a diverse gene cassette array with 16% novel gene cassettes that play a vital role in pathogenesis, transportation, biosynthesis, and AMR. Most resistance-related gene cassettes were associated with the genes encoding resistance to quaternary ammonium compound (QAC) and aminoglycosides. This study highlights the significant differences in the abundance of integrons among various agricultural soils and offers deep insight into the pools of gene cassettes that play a key role in the dissemination of integrons and AMR.

BACTERIAL PROFILES OF KARVACHAR HOT SPRING IDENTIFIED BY COMBINATION OF DIFFERENT MOLECULAR APPROACHES

Molecular techniques, including denaturing gradient gel electrophoresis (DGGE), 16S rRNA genes clone library construction and metagenomic analysis, were used to describe the bacterial composition of the Karvachar geothermal spring. It was shown the predominance of bacteria belonging to the phyla Proteobacteria, Bacteroidetes, Firmicutes and Cyanobacteria in the studied spring. Representatives of phylum Firmicutes were not detected in the clone library, while DGGE profiling and metagenome analysis confirmed the presence of Firmicutes as one of the major components in the bacterial community.

A mixed consortium of methanotrophic archaea and bacteria boosts methane-dependent selenate reduction

Though methane-based selenate reduction has been reported, neither the selenate load nor the removal rate could satisfy practical applications, thus limiting this technique to bio-remediate selenate pollution. In the present study, using a membrane biofilm batch reactor (MBBR), we successfully enriched a consortium performing methane-dependent selenate reduction, with enhanced reduction rates from 16.1 to 28.9 μM-day−1 under a comparable Se concentration to industrial wastewaters (i.e., ~500 μM). During active reduction, 16S rRNA gene copies of Archaea and Bacteria were both increased more than one order of magnitude. Clone library construction and high-throughput sequencing indicated that Methanosarcina and Methylocystis were the only methane-oxidizing microorganisms. The presence of 20 mM bromoethanesulphonate or 0.15 mM acetylene both significantly, but not completely, inhibited methane-dependent selenate reduction, indicating the concurrent contributions of methanotrophic archaea and bacteria. Fluorescence in situ hybridization (FISH) revealed that archaea directly adhered to the surface of the membrane while bacteria were in the outer layer, together forming the mature biofilm. This study highlights the crucial role of both methanotrophic archaea and bacteria in methane-dependent selenate reduction, and lays foundations in applying methane to bio-remediate practical selenate pollution.

Functional diversity and abundance of nitrogen cycle-related genes in paddy soil

The nitrogen cycle and the associated microbes play an important role in natural ecosystems, including terrestrial habitats; they also have a major effect on climate change. The aim of this study was to explore microbial communities in rice paddy soil by detecting and quantifying some key functional genes involved in the nitrogen cycle using molecular techniques such as conventional polymerase chain reaction (PCR), clone library construction, sequencing, phylogenetic analysis, and real-time PCR. The genes analyzed were as follows: nitrogenase reductase gene (nifH), hydrazine synthase gene (hzsA), nitrous oxide reductase gene (nosZ), copper-containing (nirK) and cytochrome cd1-containing (nirS) nitrite reductase genes, nitrite oxidoreductase gene (nxrB), and ammonium monooxygenase gene (amoA). The sequence assessment using the clone library targeting these genes revealed high diversity and dominance of bacterial communities. Furthermore, real-time PCR using SYBR green dye and some primers specific for each gene revealed the high abundance of nxrB (4.1 × 109 ± 0.4 × 109 copies g−1 soil) and low abundance of hzsA (4.0 × 105 ± 1.1 × 105 copies g−1 soil). The findings of our study will be useful to explore microbial communities in terrestrial habitats, such as agricultural paddy fields.

Identifying the core bacterial microbiome of hydrocarbon degradation and a shift of dominant methanogenesis pathways in the oil and aqueous phases of petroleum reservoirs of different temperatures from China

Abstract. Microorganisms in petroleum reservoirs play significant roles in hydrocarbon degradation, and through the terminal electron-accepting process of methanogenesis, they also contribute to microbially enhanced oil recovery (MEOR) worldwide, with great economic and environmental benefits. Here, a molecular investigation, using the 16S rRNA and mcrA gene profiles based on MiSeq sequencing and clone library construction methods, was conducted on oil and water (aqueous) phases of samples of high (82–88 ∘C), moderate (45–63 ∘C), and low temperatures (21–32 ∘C) from seven petroleum reservoirs in China. A core bacterial microbiome with a small proportion of shared operational taxonomic unit (OTU) values, but a high proportion of sequences among all reservoirs was discovered, including aerobic degraders, sulfate- and nitrate-reducing bacteria, fermentative bacteria, and sulfur-oxidizing bacteria distributed mainly in Proteobacteria, Bacteroidetes, Deferribacteres, Deinococcus–Thermus, Firmicutes, Spirochaetes, and Thermotogae. Their prevalence in the previously reported petroleum reservoirs and successive enrichment cultures suggests their common roles and functions involved in aliphatic and aromatic hydrocarbon degradation. The methanogenic process generally shifts from the dominant hydrogenotrophic pathway in the aqueous phase to the acetoclastic pathway in the oil phase in high-temperature reservoirs, but the opposite was true for low-temperature reservoirs. No difference was detected between the two phases in moderate temperature reservoirs. Physicochemical factors, including pH; temperature; phase conditions; and nitrate, Mn2+, and Mg2+ concentrations were the main factors correlated to the microbial compositional and functional profiles significantly. Linear discriminant analysis (LDA) effect size (LEfSe) analysis shows distribution differences of microbial groups towards pH, temperature, and the oil and aqueous phases. Using the software Tax4Fun for functional profiling indicated functional metabolism differences between the two phases, including amino acids, hydrocarbons in the oil phase, and carbohydrates in the aqueous phase.

Fungal Succession During the Decomposition of Ectomycorrhizal Fine Roots.

Ectomycorrhizal (ECM) fine roots account for a substantial proportion of forest production and their decomposition releases large amounts of nutrients to the soil ecosystem. However, little is known about the fungi involved in ECM decomposition, including assemblages of fungal saprotrophs, endophytes, and the ECM fungi themselves. To follow fungal succession during the degradation of senescing fine roots, understory seedlings of Abies balsamea and Picea rubens at two sites in the Acadian forest of Nova Scotia were either severed at the root collar or left as controls. Root systems were collected sequentially over two growing seasons and assessed for fine root loss and ECM mantle integrity. ECM were identified by ITS-PCR and grouped into broad morphological categories. Fungal communities colonizing the senescing fine roots were also monitored by systematically constructing clone libraries over the course of the experiment. ECM with cottony, weakly pigmented mantles (e.g., Cortinarius) degraded within the first year. Those with cottony, but intensely pigmented mantles (Piloderma), and smooth mantles with weak pigmentation (Russulaceae) degraded more slowly. Smooth, melanized ECM (Cenococcum and Tomentella) generally maintained integrity over the course of the experiment. Rates of fine root loss and changes in ECM mantle integrity were positively correlated with soil temperature. ECM DNA was detected throughout the experiment, and was not replaced by that of saprotrophic species during the two seasons sampled. However, fungal root endophytes (e.g., Helotiaceae) initially increased in abundance and then decreased as mantles degraded, suggesting a possible role in ECM decomposition.

Vertical Variation of Nitrogen Fixers and Ammonia Oxidizers along a Sediment Profile in the Dajiuhu Peatland, Central China

To investigate the vertical variation of microbial functional groups particular nitrogen fixers and ammonia oxidizers, sediment samples from a 155 cm deep peat profile were collected from the acidic Dajiuhu Peatland and subsequently subjected to clone library construction and quantification. Results showed that nifH gene abundance varied between 105–108 copies per gram dry sediment and reduced gradually with depth. The abundances of ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) abundance were generally comparable in each sample. More AOA was observed with a depth ≤55 cm, whereas AOB was more abundant with a depth >55 cm. Phylogenetically nifH could be divided into 94 OTUs which mainly affiliated with α-Proteobacteria. AOA were affiliated with Nitrosotalea from Group 1.1a associated (nearly 90%) and Nitrososphaera from Group I.1b. All AOB belonged to Nitrosospira. Notably, DJH11 with the depth of 50–55 cm was observed to have the highest abundance and the highest diversity of nitrogen fixers and ammonia oxidizers among all the samples. Pearson’s correlation analysis showed a positive relationship between water content and pH with the nifH gene abundance. Our results offer the first insight about the microbial community composition and diversity involved in nitrogen cycles in the Dajiuhu Peatland.

Spatiotemporal differences in the arbuscular mycorrhizal fungi communities in soil and roots in response to long-term organic compost inputs in an intensive agricultural cropping system on the North China Plain

Fertilizer management is important for minimizing nutrient losses from intensive agroecosystems. An increasing amount of organic fertilizer has been applied to overcome the problems associated with mineral fertilizers. Thus, an understanding of the diversity and community structure changes in arbuscular mycorrhizal fungi (AMF) in response to long-term organic fertilizer management can be potentially significant in the development of nutrient management strategies. Here, a study was conducted to investigate the vertical distribution of AMF in a calcareous field and the temporal structure of AMF in maize roots with different levels of continuous fertilization over a 13-year period. T-RFLP and clone library construction were used to investigate AMF community in this study. Canonical correspondence analysis was performed to determine the significance of environmental variable that may affect the AMF community composition. Our results showed that the Shannon-Weiner and evenness indexes of soil AMF community decreased, while AMF richness was not significantly affected. Organic compost application reduced root colonization, while the negative influence of conventional inorganic fertilization was minor. The effect was significant at 13 leaf collar stage of maize. Crop phenology especially growth stages might override fertilizer supply in determining the community composition of active root inhabiting AM fungi. Significant differences in the community structure of soil AMF were observed between control and organic compost treatments in surface soil, and the community shift was primarily attributable to soil organic matter and nutrient contents (total nitrogen and carbon, Olsen-P, and exchangeable K). Vertical distribution of AMF was significantly related to soil electrical conductivity and pH values. Our results indicated that AMF community assemblage was complex and dependent on fertilization-mediated changes in soil properties, soil depth, and crop phenology. The modification of AMF communities by fertilization may have great impact on soil health and ecosystem services in intensive agroecosystems.

Retraction Note to: An assessment of transgenomics as a tool for gene discovery in Populus euphratica Oliv.

This article (Zhou et al. 2018) has been retracted by the authors because the sequence BIBAC 002A111F06 was incorrectly assigned to the wrong bacterial species. The BIBAC 002A111F06 sequence (GenBank Accession KC129717) reported in the paper was attributed to Populus euphratica Oliv. The BLAST search of this KC129717 sequence against the nr database at NCBI showed that it has very high similarity to a genomic sequence from the gram-negative bacteria Stenotrophomonas maltophilia. The bacterium associates with Populus euphratica Oliv. and DNA isolated from Populus euphratica Oliv. for the construction of the BIBAC clone library inlcuded DNA from Stenotrophomonas maltophilia. Therefore, the phenotype of the transgenic Arabidopsis line carrying the KC129717 sequence cannot be attributed to genes from Populus euphratica Oliv. The authors apologize for the confusion and misinterpretation of our data resulting from the incorrect sequence assignment. All authors agree to this retraction.

The microbiome in the lower respiratory tract.

With the advent of new technologies evaluating the microbiome in the sample such as next-generation sequencer (NGS), current increase of an interest in understanding of the lung microbiome and its roles in lung diseases are marked. Gathering the data of bacterial flora in the lung and their changes during disease courses is unraveling the pathogenesis and the mechanism of disease progression particularly in patients with bronchial asthma, chronic obstructive pulmonary disease and infectious lung diseases. To clarify the relationship between the lung microbiome and pulmonary diseases, new information may help us to create new treatment and prevention strategies of some pulmonary diseases by controlling the lung microbiome. Using bacterial 16S ribosomal RNA gene sequence, NGS can rapidly estimate large amount of bacterial sequences in the phylum and genus levels, and some of them in species levels in a very short period of time. In addition to new information of the microbiome using NGS in the respiratory tract, other techniques using basically Sanger method in combination with the clone library construction can also be useful to identify pathogenic bacterial species with their ratio in the respiratory samples such as bacterial pneumonia, lung abscess and nontuberculous mycobacteriosis. These modalities to identify and semi-quantify bacterial burden in the respiratory tract have revealed new bacterial information in each infectious lung disease. This review describes current understanding of the lung microbiome in several representative lung diseases.

Spatiotemporal distributions and environmental drivers of diversity and community structure of nosZ-type denitrifiers and anammox bacteria in sediments of the Bohai Sea and North Yellow Sea, China

Denitrification and anammox processes are major nitrogen removal processes in coastal ecosystems. However, the spatiotemporal dynamics and driving factors of the diversity and community structure of involved functional bacteria have not been well illustrated in coastal environments, especially in human-dominated ecosystems. In this study, we investigated the distributions of denitrifiers and anammox bacteria in the eutrophic Bohai Sea and the northern Yellow Sea of China in May and November of 2012 by constructing clone libraries employing nosZ and 16S rRNA gene biomarkers. The diversity of nosZ-denitrifier was much higher at the coastal sites compared with the central sites, but not significant among basins or seasons. Alphaproteobacteria were predominant and prevalent in the sediments, whereas Betaproteobacteria primarily occurred at the site near the Huanghe (Yellow) River estuary. Anammox bacteria Candidatus Scalindua was predominant in the sediments, and besides, Candidatus Brocadia and Candidatus Kuenenia were also detected at the site near the Huanghe River estuary that received strong riverine and anthropogenic impacts. Salinity was the most important in structuring communities of nosZ-denitrifier and anammox bacteria. Additionally, anthropogenic perturbations (e.g. nitrogen overloading and consequent high primary productivity, and heavy metal discharges) contributed significantly to shaping community structures of denitrifier and anammox bacteria, suggesting that anthropogenic activities would influence and even change the ecological function of coastal ecosystems.

CLONING OF GENES ENCODING TRANSMEMBRANE PROTEINS AND PROTEINS RESPONSIBLE FOR AFRICAN SWINE FEVER VIRUS VIRULENCE

Results of cloning X69R, A179L, E248R, I215L and DP96R genes of ASF virus Krasnodar 07/17 isolate and analysis of their nucleotide sequences are presented. Obtained clones were added to the previously constructed clone library comprising clones of 8 genes of Krasnodar 06/12 isolate. Clones containing X69R, A179L, E248R, I215L and DP96R genes of ASF virus Krasnodar 07/17 isolate will be used for recombinant protein obtaining and testing for their effect on in vitro virus reproduction and their role in the virus infectivity, level of clinical manifestations and virulence. Prokaryotic vector, pJET1.2/ blunt, was used. Thus, the clone library available at the FGBI “ARRIAH” Reference Laboratory for African swine fever was supplemented by pJET1.2-X69R, pJET1.2-A179L, pJET1.2-E248R, pJET1.2-I215L and pJET1.2-DP96R plasmid constructions containing 5 genes of ASF virus Krasnodar 07/17 isolate. Proportion of cloned virus genes was 3.01% of Krasnodar 07/17 isolate genome, hence, total amount of the clone library has reached 7.82%.

CLONING OF GENES ENCODING TRANSMEMBRANE PROTEINS AND PROTEINS RESPONSIBLE FOR AFRICAN SWINE FEVER VIRUS VIRULENCE

Results of cloning X69R, A179L, E248R, I215L and DP96R genes of ASF virus Krasnodar 07/17 isolate and analysis of their nucleotide sequences are presented. Obtained clones were added to the previously constructed clone library comprising clones of 8 genes of Krasnodar 06/12 isolate. Clones containing X69R, A179L, E248R, I215L and DP96R genes of ASF virus Krasnodar 07/17 isolate will be used for recombinant protein obtaining and testing for their effect on in vitro virus reproduction and their role in the virus infectivity, level of clinical manifestations and virulence. Prokaryotic vector, pJET1.2/ blunt, was used. Thus, the clone library available at the FGBI “ARRIAH” Reference Laboratory for African swine fever was supplemented by pJET1.2-X69R, pJET1.2-A179L, pJET1.2-E248R, pJET1.2-I215L and pJET1.2-DP96R plasmid constructions containing 5 genes of ASF virus Krasnodar 07/17 isolate. Proportion of cloned virus genes was 3.01% of Krasnodar 07/17 isolate genome, hence, total amount of the clone library has reached 7.82%.