rRNA Operons Research Articles

Complete genome sequence of Roseivivax marinus strain TCYB24 with quorum sensing system reveal the adaptive mechanism against deep-sea hydrothermal environment.

Roseivivax marinus strain TCYB24 is a rod-shaped bacterium of Rhodobacteraceae isolated from the gill of deep-sea mussel Bathymodiolus marisindicus which collected from the Tiancheng hydrothermal vent under depth of 2700m on the southwest Indian ridge. In our previous study, the strain TCYB24 was proved to produce quorum sensing signal of N-Acyl-homoserine lactones (AHLs) and form biofilm. In order to determine its adaptive mechanism against the extreme environment of deep-sea hydrothermal vents, the whole genome was sequenced by high-throughput Illumina tag sequencing. The results show the whole genome consists of one circular chromosome and eight circular plasmids, with a total length of 4.60Mb (G+C content of 67.4%), 4338 open reading frames, 46 tRNAs and 6 rRNA operons. According to the genome-wide functional annotation, numbers of heavy metal resistance, high pressure and cold adapting related genes were found. In addition, genes about exopolysaccharide (EPS) biosynthesis and secretion and biofilm formation, which facilitate bacteria to resist extreme environments, were identified. Intriguingly, a pair of RaiI/R-type quorum sensing system was discovered firstly in the bacterium isolated from hydrothermal environment. The results may help to understand genetic underpinning of extreme environmental adaptation mechanism of bacteria in deep-sea hydrothermal area.

Evolutionary Dynamics and Lateral Gene Transfer in Raphidophyceae Plastid Genomes.

The Raphidophyceae is an ecologically important eukaryotic lineage of primary producers and predators that inhabit marine and freshwater environments worldwide. These organisms are of great evolutionary interest because their plastids are the product of eukaryote-eukaryote endosymbiosis. To obtain deeper insight into the evolutionary history of raphidophycean plastids, we sequenced and analyzed the plastid genomes of three freshwater and three marine species. Our comparison of these genomes, together with the previously reported plastid genome of Heterosigma akashiwo, revealed unexpected variability in genome structure. Unlike the genomes of other analyzed species, the plastid genome of Gonyostomum semen was found to contain only a single rRNA operon, presumably due to the loss of genes from the inverted repeat (IR) region found in most plastid genomes. In contrast, the marine species Fibrocapsa japonica contains the largest IR region and overall plastid genome for any raphidophyte examined thus far, mainly due to the presence of four large gene-poor regions and foreign DNA. Two plastid genes, tyrC in F. japonica and He. akashiwo and serC in F. japonica, appear to have arisen via lateral gene transfer (LGT) from diatoms, and several raphidophyte open reading frames are demonstrably homologous to sequences in diatom plasmids and plastid genomes. A group II intron in the F. japonica psbB gene also appears to be derived by LGT. Our results provide important insights into the evolutionary history of raphidophyte plastid genomes via LGT from the plastids and plasmid DNAs of diatoms.

Complete Genome Sequence Resource for Xanthomonas translucens pv. undulosa MAI5034, a Wheat Pathogen from Uruguay.

Complete Genome Sequence Resource for Xanthomonas translucens pv. undulosa MAI5034, a Wheat Pathogen from Uruguay.

RRNA operon multiplicity as a bacterial genome stability insurance policy.

Quick growth restart after upon encountering favourable environmental conditions is a major fitness contributor in natural environment. It is widely assumed that the time required to restart growth after nutritional upshift is determined by how long it takes for cells to synthesize enough ribosomes to produce the proteins required to reinitiate growth. Here we show that a reduction in the capacity to synthesize ribosomes by reducing number of ribosomal RNA (rRNA) operons (rrn) causes a longer transition from stationary phase to growth of Escherichia coli primarily due to high mortality rates. Cell death results from DNA replication blockage and massive DNA breakage at the sites of the remaining rrn operons that become overloaded with RNA polymerases (RNAPs). Mortality rates and growth restart duration can be reduced by preventing R-loop formation and improving DNA repair capacity. The same molecular mechanisms determine the duration of the recovery phase after ribosome-damaging stresses, such as antibiotics, exposure to bile salts or high temperature. Our study therefore suggests that a major function of rrn operon multiplicity is to ensure that individual rrn operons are not saturated by RNAPs, which can result in catastrophic chromosome replication failure and cell death during adaptation to environmental fluctuations.

Typical structure of rRNA coding genes in diplonemids points to two independent origins of the bizarre rDNA structures of euglenozoans

BackgroundMembers of Euglenozoa (Discoba) are known for unorthodox rDNA organization. In Euglenida rDNA is located on extrachromosomal circular DNA. In Kinetoplastea and Euglenida the core of the large ribosomal subunit, typically formed by the 28S rRNA, consists of several smaller rRNAs. They are the result of the presence of additional internal transcribed spacers (ITSs) in the rDNA. Diplonemea is the third of the main groups of Euglenozoa and its members are known to be among the most abundant and diverse protists in the oceans. Despite that, the rRNA of only one diplonemid species, Diplonema papillatum, has been examined so far and found to exhibit continuous 28S rRNA. Currently, the rDNA organization has not been researched for any diplonemid. Herein we investigate the structure of rRNA genes in classical (Diplonemidae) and deep-sea diplonemids (Eupelagonemidae), representing the majority of known diplonemid diversity. The results fill the gap in knowledge about diplonemid rDNA and allow better understanding of the evolution of the fragmented structure of the rDNA in Euglenozoa.ResultsWe used available genomic (culture and single-cell) sequencing data to assemble complete or almost complete rRNA operons for three classical and six deep-sea diplonemids. The rDNA sequences acquired for several euglenids and kinetoplastids were used to provide the background for the analysis. In all nine diplonemids, 28S rRNA seems to be contiguous, with no additional ITSs detected. Similarly, no additional ITSs were detected in basal prokinetoplastids. However, we identified five additional ITSs in the 28S rRNA of all analysed metakinetoplastids, and up to twelve in euglenids. Only three of these share positions, and they cannot be traced back to their common ancestor.ConclusionsPresented results indicate that independent origin of additional ITSs in euglenids and kinetoplastids seems to be the most likely. The reason for such unmatched fragmentation remains unknown, but for some reason euglenozoan ribosomes appear to be prone to 28S rRNA fragmentation.

Short- and long-read metabarcoding of the eukaryotic rRNA operon: Evaluation of primers and comparison to shotgun metagenomics sequencing.

High-throughput sequencing-based analysis of microbial diversity has evolved vastly over the last decade. Currently, the go-to method for studying microbial eukaryotes is short-read metabarcoding of variable regions of the 18S rRNA gene with <500bp amplicons. However, there is a growing interest in applying long-read sequencing of amplicons covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is representative. Here, we designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. The primers were evaluated in silico along with published primers on reference sequence databases and marine metagenomics data sets. We further evaluated a subset of the primers for short- and long-read sequencing on environmental samples in vitro and compared the obtained community profile with primer-unbiased metagenomic sequencing. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Fewer differences were observed between the long-read primer pairs. The long-read amplicons and ITS1 alone provided higher taxonomic resolution than V4. Together, our results represent a reference and guide for selection of robust primers for research on and environmental monitoring of microbial eukaryotes.

Complete genome sequence of Rhodoferax sp. PAMC 29310 from a marine sediment of the East Siberian Sea.

Rhodoferax sp. PAMC 29310 was isolated from a surface marine sediment of the East Siberian Sea, Arctic. Whole-genome sequencing of the strain Rhodoferax sp. PAMC 29310 was achieved using PacBio RS II and Illumina platform. The resulting complete genome comprised of 4,593,249 base pairs (G+C content of 58.0%) with a single chromosome, 4546 protein-coding genes, 57 tRNAs and 6 rRNA operons. A complete set of genes encoding the enzymes of glycolysis and citric acid cycle were identified. No genes encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and nitrogenase reductase (nif) were present indicating that strain PAMC 29310 is not capable of fixing of carbon and nitrogen. PAMC 29310 genome contains genes for dissimilatory and assimilatory nitrate reduction. Gene encoding choline dehydrogenase enzyme which functions at the first step in the synthesis of betaine, one of the most effective osmoprotectants, was detected. In particular, among the genomes of the genus Rhodoferax strains, gene encoding nitrite reductase (nirK), which reduces nitrite to nitric oxide and tetA gene encoding tetracycline resistance protein involved in the resistance to tetracycline were identified only in the genome of Rhodoferax sp. PAMC 29310. As the first genome from the strain which was isolated from marine sediment in the genus Rhodoferax, investigation of physiological characteristics based on the complete genome sequences will help understand the adaptation of Rhodoferax sp. PAMC 29310 in the marine sediment.

Complete genome of Nocardioides aquaticus KCTC 9944T isolated from meromictic and hypersaline Ekho Lake, Antarctica.

Nocardioides aquaticus KCTC 9944T is an aerobic, non-motile, Gram-positive, psychrotolerant, non-spore-forming bacterium isolated from the surface water of Ekho Lake in the Vestfold Hills, East Antarctica. This meromictic lake separated from Antarctic seawater thousands of years ago exhibits steep gradients of salinity and temperature in the upper layer of the water column. The cells of N. aquaticus thriving in Ekho Lake are able to grow in wide ranges of temperature (3 to 43.5°C) and salinity (0 to 15% NaCl). Here, we sequenced the complete genome of N. aquaticus KCTC 9944T, aiming to better understand the adaptation of this bacterium to the strong environmental gradients at the molecular level. The genome consists of 4,580,814bp (G+C content of 73.2%) with a single chromosome, 4432 protein-coding genes, 51 tRNAs and 2 rRNA operons. The genome possesses genes for the Entner-Doudoroff pathway, photoheterotrophy, the conversion of acetate to acetyl-CoA, gluconeogenesis, and energy storage that are all advantageous to oligotrophic bacteria. The presence of genes involved in osmotic balance, fatty acid desaturation, cold and heat shock responses, and the oxygen affinities of respiratory oxidases are likely associated with high tolerance to strong gradients of salinity, temperature and oxygen concentration.

Microbial Identification Using rRNA Operon Region: Database and Tool for Metataxonomics with Long-Read Sequence.

ABSTRACTRecent development of long-read sequencing platforms has enabled researchers to explore bacterial community structure through analysis of full-length 16S rRNA gene (∼1,500 bp) or 16S-ITS-23S rRNA operon region (∼4,300 bp), resulting in higher taxonomic resolution than short-read sequencing platforms. Despite the potential of long-read sequencing in metagenomics, resources and protocols for this technology are scarce. Here, we describe MIrROR, the database and analysis tool for metataxonomics using the bacterial 16S-ITS-23S rRNA operon region. We collected 16S-ITS-23S rRNA operon sequences extracted from bacterial genomes from NCBI GenBank and performed curation. A total of 97,781 16S-ITS-23S rRNA operon sequences covering 9,485 species from 43,653 genomes were obtained. For user convenience, we provide an analysis tool based on a mapping strategy that can be used for taxonomic profiling with MIrROR database. To benchmark MIrROR, we compared performance against publicly available databases and tool with mock communities and simulated data sets. Our platform showed promising results in terms of the number of species covered and the accuracy of classification. To encourage active 16S-ITS-23S rRNA operon analysis in the field, BLAST function and taxonomic profiling results with 16S-ITS-23S rRNA operon studies, which have been reported as BioProject on NCBI are provided. MIrROR (http://mirror.egnome.co.kr/) will be a useful platform for researchers who want to perform high-resolution metagenome analysis with a cost-effective sequencer such as MinION from Oxford Nanopore Technologies.IMPORTANCE Metabarcoding is a powerful tool to investigate community diversity in an economic and efficient way by amplifying a specific gene marker region. With the advancement of long-read sequencing technologies, the field of metabarcoding has entered a new phase. The technologies have brought a need for development in several areas, including new markers that long-read can cover, database for the markers, tools that reflect long-read characteristics, and compatibility with downstream analysis tools. By constructing MIrROR, we met the need for a database and tools for the 16S-ITS-23S rRNA operon region, which has recently been shown to have sufficient resolution at the species level. Bacterial community analysis using the 16S-ITS-23S rRNA operon region with MIrROR will provide new insights from various research fields.

Complete Genome Sequence of Xanthomonas campestris pv. campestris SB80, a Race 4 Strain Isolated from White Head Cabbage in Turkey.

ABSTRACTHere, we report the complete genome sequence of the race 4 strain Xanthomonas campestris pv. campestris SB80, which was isolated from a symptomatic white head cabbage leaf in Samsun Province, Turkey, in 2019. The genome consists of a circular chromosome (5,129,762 bp) with a G+C content of 64.98%, for which 4,159 putative protein-coding genes, 2 rRNA operons, 54 tRNAs, and 86 noncoding RNAs (ncRNAs) were predicted.

Complete genome sequence of Polaribacter sejongensis NJDZ03 exhibiting diverse macroalgal polysaccharide-degrading activity.

A macroalgal polysaccharide-degrading bacterial strain, Polaribacter sejongensis NJDZ03, was isolated from the surface of a species of Desmarestia, a seaweed genus endemic to Antarctica. To explore the mechanism underlying the multiple polysaccharide-degrading abilities of this strain, we sequenced and analyzed its complete genome. We found that the genome comprises a 4,078,668bp circular chromosome containing 3484 coding genes, including 18 rRNA operons whose genes are arranged in the order of 16S-23S-5S rRNA. Gene annotation revealed the existence of three putative polysaccharide utilization loci (PULs) consisting of several agarase, carrangeenase, and alginate lyase genes, repectively. These PULs are likely responsible for the strong agar-, alginate-, and carrageenan-degrading capabilities of P. sejongensis NJDZ03, especially its ability to degrade diverse carrageenans, including κ-carrageenan, τ-carrageenan, and λ-carrageenan. Our findings should provide new insights into the carbon cycle of the Antarctic oceanic ecosystem.

A ribosomal operon database and MegaBLAST settings for strain-level resolution of microbiomes.

Current methods to characterize microbial communities generally employ sequencing of the 16S rRNA gene (<500bp) with high accuracy (∼99%) but limited phylogenetic resolution. However, long-read sequencing now allows for the profiling of near-full-length ribosomal operons (16S-ITS-23S rRNA genes) on platforms such as the Oxford Nanopore MinION. Here, we describe an rRNA operon database with >300 ,000entries, representing >10 ,000prokaryotic species and ∼ 150, 000 strains. Additionally, BLAST parameters were identified for strain-level resolution using in silico mutated, mock rRNA operon sequences (70-95% identity) from four bacterial phyla and two members of the Euryarchaeota, mimicking MinION reads. MegaBLAST settings were determined that required <3s per read on a Mac Mini with strain-level resolution for sequences with >84%identity. These settings were tested on rRNA operon libraries from the human respiratory tract, farm/forest soils and marine sponges ( n = 1, 322, 818 reads for all sample sets). Most rRNA operon reads in this data set yielded best BLAST hits (95± 8%). However, only 38-82% of library reads were compatible with strain-level resolution, reflecting the dominance of human/biomedical-associated prokaryotic entries in the database. Since the MinION and the Mac Mini are both portable, this study demonstrates the possibility of rapid strain-level microbiome analysis in the field.

In-Depth Analysis of Bacillus anthracis 16S rRNA Genes and Transcripts Reveals Intra- and Intergenomic Diversity and Facilitates Anthrax Detection.

ABSTRACTAnalysis of 16S rRNA (rRNA) genes provides a central means of taxonomic classification of bacterial species. Based on presumed sequence identity among species of the Bacillus cereus sensu lato group, the 16S rRNA genes of B. anthracis have been considered unsuitable for diagnosis of the anthrax pathogen. With the recent identification of a single nucleotide polymorphism in some 16S rRNA gene copies, specific identification of B. anthracis becomes feasible. Here, we designed and evaluated a set of in situ, in vitro, and in silico assays to assess the unknown 16S state of B. anthracis from different perspectives. Using a combination of digital PCR, fluorescence in situ hybridization, long-read genome sequencing, and bioinformatics, we were able to detect and quantify a unique 16S rRNA gene allele of B. anthracis (16S-BA-allele). This allele was found in all available B. anthracis genomes and may facilitate differentiation of the pathogen from any close relative. Bioinformatics analysis of 959 B. anthracis SRA data sets inferred that abundances and genomic arrangements of the 16S-BA-allele and the entire rRNA operon copy numbers differ considerably between strains. Expression ratios of 16S-BA-alleles were proportional to the respective genomic allele copy numbers. The findings and experimental tools presented here provide detailed insights into the intra- and intergenomic diversity of 16S rRNA genes and may pave the way for improved identification of B. anthracis and other pathogens with diverse rRNA operons.IMPORTANCE For severe infectious diseases, precise pathogen detection is crucial for antibiotic therapy and patient survival. Identification of Bacillus anthracis, the causative agent of the zoonosis anthrax, can be challenging when querying specific nucleotide sequences such as in small subunit rRNA (16S rRNA) genes, which are commonly used for typing of bacteria. This study analyzed on a broad genomic scale a cryptic and hitherto underappreciated allelic variant of the bacterium’s 16S rRNA genes and their transcripts using a set of in situ, in vitro, and in silico assays and found significant intra- and intergenomic heterogeneity in the distribution of the allele and overall rRNA operon copy numbers. This allelic variation was uniquely species specific, which enabled sensitive pathogen detection on both DNA and transcript levels. The methodology used here is likely also applicable to other pathogens that are otherwise difficult to discriminate from their less harmful relatives.

Complete Genome Sequence of Curtobacterium sp. Strain TXMA1, Isolated from a Grapevine in Texas, USA.

ABSTRACTThe genome of Curtobacterium sp. strain TXMA1, isolated from a grapevine in Texas showing leaf marginal necrosis symptoms, was sequenced. The TXMA1 genome has a 3,454,876-bp, circular chromosome with a GC content of 71.74%, 3,213 open reading frames (ORFs), 47 tRNAs, and 4 complete rRNA operons (5S, 16S, and 23S).

Natural Chromosome-Chromid Fusion across rRNA Operons in a Burkholderiaceae Bacterium.

ABSTRACTChromids (secondary chromosomes) in bacterial genomes that are present in addition to the main chromosome appear to be evolutionarily conserved in some specific bacterial groups. In rare cases among these groups, a small number of strains from Rhizobiales and Vibrionales were shown to possess a naturally fused single chromosome that was reported to have been generated through intragenomic homologous recombination between repeated sequences on the chromosome and chromid. Similar examples have never been reported in the family Burkholderiaceae, a well-documented group that conserves chromids. Here, an in-depth genomic characterization was performed on a Burkholderiaceae bacterium that was isolated from a soil bacterial consortium maintained on diesel fuel and mutagenic benzo[a]pyrene. This organism, Cupriavidus necator strain KK10, was revealed to carry a single chromosome with unexpectedly large size (>6.6 Mb), and results of comparative genomics with the genome of C. necator N-1T indicated that the single chromosome of KK10 was generated through fusion of the prototypical chromosome and chromid at the rRNA operons. This fusion hypothetically occurred through homologous recombination with a crossover between repeated rRNA operons on the chromosome and chromid. Some metabolic functions that were likely expressed from genes on the prototypical chromid region were indicated to be retained. If this phenomenon—the bacterial chromosome-chromid fusion across the rRNA operons through homologous recombination—occurs universally in prokaryotes, the multiple rRNA operons in bacterial genomes may not only contribute to the robustness of ribosome function, but also provide more opportunities for genomic rearrangements through frequent recombination.IMPORTANCE A bacterial chromosome that was naturally fused with the secondary chromosome, or “chromid,” and presented as an unexpectedly large single replicon was discovered in the genome of Cupriavidus necator strain KK10, a biotechnologically useful member of the family Burkholderiaceae. Although Burkholderiaceae is a well-documented group that conserves chromids in their genomes, this chromosomal fusion event has not been previously reported for this family. This fusion has hypothetically occurred through intragenomic homologous recombination between repeated rRNA operons and, if so, provides novel insight into the potential of multiple rRNA operons in bacterial genomes to lead to chromosome-chromid fusion. The harsh conditions under which strain KK10 was maintained—a genotoxic hydrocarbon-enriched milieu—may have provided this genotype with a niche in which to survive.

Depth effects on bacterial community assembly processes in paddy soils

Bacterial communities in soil play a key role in carbon (C) and nutrient cycling. Unravelling how bacterial community assemble and distribute with soil depth is a prerequisite for understanding microbial functions, nutrient cycling and management. Twenty-six rice fields in a typical red soil area in a wet subtropical climate were sampled in the topsoil (0–10 and 10–20 cm) and subsoil (20–40 cm). Physico-chemical soil properties, quantitative fluorescence PCR and high-throughput sequencing were used to analyse the V4 region of 16S rDNA. The rRNA operon copy number and alpha diversity decreased continuously with soil depth because of reduced access to carbon, energy, oxygen and nutrients. The relative abundance of the dominant phyla Proteobacteria and Actinobacteria decreased with increasing soil depth, whereas the opposite trend was observed for the phylum Nitrospirae. The interaction intensity between taxa increased with depth, as limited carbon and nutrients in the undisturbed subsoil lead to the cooccurrence of taxa with similar ecological niches that cooperated to reduce functional redundancy. The higher modularity of the bacterial network in the topsoil is associated with greater environmental perturbations (flooding, fertilization, etc.) to maintain the robustness of the microbial community. Bacterial community assembly processes were stochastic up to 40 cm, but ecological drift was the predominant process in the topsoil, whereas dispersal limitation was dominant in the subsoil. The contribution of abiotic factors (e.g. nutrient and iron contents) and biotic factors (taxa-taxa interactions) as well as dispersal limitations to bacterial community assembly was depth specific. Concluding, the basic principles of bacterial community assembly were evaluated for the first time for a broad range of paddy soils.

Characterization of genomic, physiological, and probiotic features Lactiplantibacillus plantarum DY46 strain isolated from traditional lactic acid fermented shalgam beverage

Lactiplantibacillus plantarum is a significant probiotic where it could be found in ubiquitous niches. In this study, a new Lb. plantarum strain DY46 was isolated from a traditional lactic-acid-fermented beverage called shalgam. The whole genome of the DY46 was sequenced and obtained sequences were assembled into a 3.32 Mb draft genome using PATRIC (3.6.8.). The DY46 genome consists of a single circular chromosome of 3,332,827 bp that is predicted to carry 3219 genes, including 61 tRNA genes, 2 rRNA operons. The genome has a GC content of 44.3% includes 98 predicted pseudogenes, 25 complete or partial transposases and 3 intact prophages. The genes encoding enzymes related in the intact EMP (Embden–Meyerhof–Parnas) and PK (phosphoketolase) pathways were predicted using BlastKOALA which is an indicator of having facultative heterofermentative pathways. DY46 genome also predicted to carry genes of Pln E, Pln F and Pln K showing the antimicrobial potential of this bacterium which can be linked to in vitro antagonism tests that DY46 can inhibit S.enterica sv. Typhimurium ATCC14028, K. pneumonie ATCC13883, and P. vulgaris ATCC8427. Moreover, it is determined that all resistome found in its genome were intrinsically originated and the strain was found to be tolerant to acid and bile concentrations by mimicking human gastrointestinal conditions. In conclusion, L. plantarum DY46 is a promising bacterium that appears to have certain probiotic properties, confirmed by “in vitro” and “in silico” analyses, and is a potential dietary supplement candidate that may provide functional benefits to the host.

Molecular Characterization of Fungi Associated with Dump Site Soil

Aims: A study was conducted to identify fungal species isolated from dumpsite soil in University of Port Harcourt using molecular techniques.&#x0D; Methodology: Molecular methods for determining the species of a fungus based on the amplification and sequencing of the internal subscribed spacer (ITS) region of the fungal rRNA operon using molecular markers was applied. Soil sample was collected from a dumpsite in the University of Port Harcourt, Rivers State, Nigeria. Isolation of fungi associated with the dumpsite soil was carried out using spread plate method. Fungal genomic DNA was extracted using Quick-DNA Fungal/Bacterial Miniprep kit. The ITS1-2 gene of the isolates was amplified by Polymerase Chain Reaction (PCR) using the primer pair; ITS4 and ITS5.&#x0D; Results: The sequences of the amplified ITS region were blasted against known sequences on the National Centre for Biotechnology Information (NCBI) database. Nucleotide sequence analysis revealed the species identity of the fungal isolates to be: Aspergillus fumigatus, Trichoderma harzianum, Aspergillus felis, Aspergillus templicola, Aspergillus flavipes, Aspergillus fumigatus and Cunninghamella binariae. Phylogenetic analysis was carried out to ascertain the relationship between the isolates and other closely-related isolates on GenBank. Isolates 2 (Trichoderma harzianum) and 7 (Cunninghamella binariae), 3 (Aspergillus felis) and 6 (Aspergillus fumigatus), and 4 (Aspergillus templicola) and 5 (Aspergillus flavipes) were found to be more closely related to each other.&#x0D; Conclusion: The molecular techniques employed successfully identified the organisms to the species level as these techniques are based on the genetic constitution of organisms. The result obtained from this study will complement the information on the fungal organisms associated with dumpsite soil.

Characterization of Microplastic-Associated Biofilm Development along a Freshwater-Estuarine Gradient.

Microplastic contamination is an increasing concern worldwide. Biofilms rapidly develop on surfaces in aquatic habitats, but the processes of biofilm formation and variation in bacterial community succession on different microplastics introduced into freshwater and estuarine environments are not well understood. In this study, the biofilm bacterial communities that developed on three different types of microplastics that are prevalent in the environment, high-density polyethylene (HDPE), polyethylene terephthalate (PET), and polystyrene (PS), was investigated. Virgin microplastics were incubated in microcosms over a period of 31 days with water collected along a freshwater-estuarine gradient of the Raritan River in New Jersey. Through long-read MinION sequencing of bacterial ribosomal operons, we were able to examine biofilm bacterial communities at a species- and strain-level resolution. Results indicated that both salinity level and microplastic type impacted biofilm formation and promoted colonization by distinct microbial communities. Limnobacter thiooxidans was found to be one of the most abundant microplastics colonizing-bacteria, and it is hypothesized that different types of microplastics could select for different strains. Our findings indicate that multiple groups of highly similar L. thiooxidans rRNA operons could be discerned within the community profiles. Phylogenetic reconstruction further established that various Linmobacter species uniquely colonized the different microplastics from the different sampling sites. Our findings indicate that microplastics support abundant and diverse bacterial communities and that the various types of microplastics can influence how different bacterial biofilms develop, which may have ecological impacts on aquatic ecosystems.

Development and validation of a positive plasmid control for detection of chlamydia genetic material in real-time polymerase chain reaction

The studies was devoted to the construction and testing of a plasmid positive control for real-time polymerase chain reaction to detect the chlamydia genome. A 142-bp region of 16s–23s rRNA operon of the chlamydia was cloned and ligated to the open plasmid vector pTZ19R, and competent E. coli cells were transformed with the resulting construct. The presence of the insert was monitored by ampicillin selection of clones and by PCR. The effectiveness of the application of the obtained structure, the copy number of which was equal to 7.65×1010 DNA molecules per μl, was proven by establishing the correlation of the values of the Ct to the amount of DNA in the sample. Testing a series of multiple dilutions of a positive plasmid control with concentrations from 101 to 107 DNA copies per μl in real-time PCR, a regression line was constructed (R2 = 0.993)