The influence of primer choice on archaeal phylogenetic analyses based on 16S rRNA gene PCR

Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Impact of Helicobacter pylori infection on fluid duodenal microbial community structure and microbial metabolic pathways

Background The bioactivities of commensal duodenal microbiota greatly influence the biofunction of hosts. We investigated the role of Helicobacter pylori infection in extra-gastroduodenal diseases by determining the impact of H. pylori infection on the duodenal microbiota. We sequenced 16 S rRNA genes in samples aspirated from the descending duodenum of 47 (male, 20; female, 27) individuals who were screened for gastric cancer. Samples were analysed using 16 S rRNA gene amplicon sequencing, and the LEFSe and Kyoto Encyclopaedia of Genes and Genomes methods were used to determine whether the duodenal microflora and microbial biofunctions were affected using H. pylori infection. Results Thirteen and 34 participants tested positive and negative for H. pylori, respectively. We identified 1,404 bacterial operational taxonomic units from 23 phyla and 253 genera. H. pylori infection changed the relative mean abundance of three phyla (Proteobacteria, Actinobacteria, and TM7) and ten genera (Neisseria, Rothia, TM7-3, Leptotrichia, Lachnospiraceae, Megasphaera, F16, Moryella, Filifactor, and Paludibacter). Microbiota features were significantly influenced in H. pylori-positive participants by 12 taxa mostly classified as Gammaproteobacteria. Microbial functional annotation revealed that H. pylori significantly affected 12 microbial metabolic pathways. Conclusions H. pylori disrupted normal bacterial communities in the duodenum and changed the biofunctions of commensal microbiota primarily by upregulating specific metabolic pathways. Such upregulation may be involved in the onset of diseases associated with H. pylori infection.

Multiple Cases of Bacterial Sequence Erroneously Incorporated Into Publicly Available Chloroplast Genomes

Public sequencing databases are invaluable resources to biological researchers, but assessing data veracity as well as the curation and maintenance of such large collections of data can be challenging. Genomes of eukaryotic organelles, such as chloroplasts and other plastids, are particularly susceptible to assembly errors and misrepresentations in these databases due to their close evolutionary relationships with bacteria, which may co-occur within the same environment, as can be the case when sequencing plants. Here, based on sequence similarities with bacterial genomes, we identified several suspicious chloroplast assemblies present in the National Institutes of Health (NIH) Reference Sequence (RefSeq) collection. Investigations into these chloroplast assemblies reveal examples of erroneous integration of bacterial sequences into chloroplast ribosomal RNA (rRNA) loci, often within the rRNA genes, presumably due to the high similarity between plastid and bacterial rRNAs. The bacterial lineages identified within the examined chloroplasts as the most likely source of contamination are either known associates of plants, or co-occur in the same environmental niches as the examined plants. Modifications to the methods used to process untargeted ‘raw’ shotgun sequencing data from whole genome sequencing efforts, such as the identification and removal of bacterial reads prior to plastome assembly, could eliminate similar errors in the future.

Bacteriohopanetetrol-<i>x</i>: constraining its application as a lipid biomarker for marine anammox using the water column oxygen gradient of the Benguela upwelling system

Interpreting lipid biomarkers in the sediment archive requires a good understanding of their application and limitations in modern systems. Recently it was discovered that marine bacteria performing anaerobic ammonium oxidation (anammox), belonging to the genus Ca. Scalindua, uniquely synthesize a stereoisomer of bacteriohopanetetrol (“BHT-x”). The ratio of BHT-x over total bacteriohopanetetrol (BHT, ubiquitously synthesized by diverse bacteria) has been suggested as a proxy for water column anoxia. As BHT has been found in sediments over 50 Myr old, BHT-x has the potential to complement and extend the sedimentary biomarker record of marine anammox, conventionally constructed using ladderane lipids. Yet, little is known about the distribution of BHT-x in relation to the distribution of ladderanes and to the genetic evidence of Ca. Scalindua in modern marine systems. Here, we investigate the distribution of BHT-x and the application of the BHT-x ratio in relation to distributions of ladderane intact polar lipids (IPLs), ladderane fatty acids (FAs) and Ca. Scalindua 16S rRNA genes in suspended particulate matter (SPM) from the water column of the Benguela upwelling system (BUS), sampled across a large oxygen gradient. In BUS SPM, high BHT-x abundances were restricted to the oxygen-deficient zone on the continental shelf (at [O2] < 45 µmol L−1, in all but one case). High BHT-x abundances co-occurred with high abundances of the Ca. Scalindua 16S rRNA gene (relative to the total number of bacterial 16S rRNA genes) and ladderane IPLs. At shelf stations with [O2] > 50 µmol L−1, the BHT-x ratio was < 0.04 (in all but one case). In apparent contradiction, ladderane FAs and low abundances of BHT and BHT-x (resulting in BHT-x ratios > 0.04) were also detected in oxygenated offshore waters ([O2] up to 180 µmol L−1), whereas ladderane IPLs were undetected. The index of ladderane lipids with five cyclobutane rings (NL5) correlates with in situ temperature. NL5-derived temperatures suggested that ladderane FAs in the offshore waters were not synthesized in situ but were transported down-slope from warmer shelf waters. Thus, in sedimentary archives of systems with known lateral organic matter transport, such as the BUS, relative BHT and BHT-x abundances should be carefully considered. In such systems, a higher BHT-x ratio may act as a safer threshold for deoxygenation and/or Ca. Scalindua presence: our results and previous studies indicate that a BHT-x ratio of ≥ 0.2 is a robust threshold for oxygen-depleted waters ([O2] < 50 µmol kg−1). In our data, ratios of ≥ 0.2 coincided with Ca. Scalindua 16S rRNA genes in all samples (n=62), except one. Lastly, when investigating in situ anammox, we highlight the importance of using ladderane IPLs over BHT-x and/or ladderane FAs; these latter compounds are more recalcitrant and may derive from transported fossil anammox bacteria remnants.

Assembly and comparative analysis of the first complete mitochondrial genome of Acer truncatum Bunge: a woody oil-tree species producing nervonic acid

Background Acer truncatum (purpleblow maple) is a woody tree species that produces seeds with high levels of valuable fatty acids (especially nervonic acid). The species is admired as a landscape plant with high developmental prospects and scientific research value. The A. truncatum chloroplast genome has recently been reported; however, the mitochondrial genome (mitogenome) is still unexplored. Results We characterized the A. truncatum mitogenome, which was assembled using reads from PacBio and Illumina sequencing platforms, performed a comparative analysis against different species of Acer. The circular mitogenome of A. truncatum has a length of 791,052 bp, with a base composition of 27.11% A, 27.21% T, 22.79% G, and 22.89% C. The A. truncatum mitogenome contains 62 genes, including 35 protein-coding genes, 23 tRNA genes and 4 rRNA genes. We also examined codon usage, sequence repeats, RNA editing and selective pressure in the A. truncatum mitogenome. To determine the evolutionary and taxonomic status of A. truncatum, we conducted a phylogenetic analysis based on the mitogenomes of A. truncatum and 25 other taxa. In addition, the gene migration from chloroplast and nuclear genomes to the mitogenome were analyzed. Finally, we developed a novel NAD1 intron indel marker for distinguishing several Acer species. Conclusions In this study, we assembled and annotated the mitogenome of A. truncatum, a woody oil-tree species producing nervonic acid. The results of our analyses provide comprehensive information on the A. truncatum mitogenome, which would facilitate evolutionary research and molecular barcoding in Acer.

Complete mitochondrial genome of Rhodeus cyanorostris (Teleostei, Cyprinidae): characterization and phylogenetic analysis

Rhodeus cyanorostris Li, Liao &amp; Arai, 2020 is a freshwater fish that is endemic to China and restricted to Chengdu City in Sichuan Province. This study is the first to sequence and characterize the complete mitochondrial genome of R. cyanorostris. The mitogenome of R. cyanorostris is 16580 bp in length, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a control region (D-loop). The base composition of the sequence is 28.5% A, 27.6% C, 26.4% T, and 17.5% G, with a bias toward A+T. The genome structure, nucleotide composition, and codon usage of the mitogenome of R. cyanorostris are consistent with those of other species of Rhodeus. To verify the molecular phylogeny of the genus Rhodeus, we provide new insights to better understand the taxonomic status of R. cyanorostris. The phylogenetic trees present four major clades based on 19 mitogenomic sequences from 16 Rhodeus species. Rhodeus cyanorostris exhibits the closest phylogenetic relationship with R. pseudosericeus, R. amarus, and R. sericeus. This study discloses the complete mitochondrial genome sequence of R. cyanorostris for the first time and provides the most comprehensive phylogenetic reconstruction of the genus Rhodeus based on whole mitochondrial genome sequences. The information obtained in this study will provide new insights for conservation, phylogenetic analysis, and evolutionary biology research.

The Complete Chloroplast Genome of Ardisia Japonica（Thunb.）Blume

Ardisia japonica（Thunb）Blume is a small shrub or sub-shrub of the genus Taurus in the Taurus family. The whole plant and root of A. japonica are used for medicinal purposes. It is a common Chinese herbal medicine and a common flower. To study its complete chloroplast genome, we collected leaves and obtained chloroplast genome information through next-generation sequencing. The results showed that the length of the genome is 155,996 bp, and the GC content ratio is 37.0%. The large single-copy region (LSC) is 86803 bp, the small single-copy region (SSC) is 18080 bp, and the inverted repeat region (IR) is 25507 bp. The chloroplast genome encodes 130 genes, including 85 protein genes, 8 rRNA genes, and 37tRNA genes. By analyzing the phylogeny of A. japonica, it is found that A. japonica and other Ardisia species are closely related.

The mitochondrial genome and phylogenetic analysis of the tick Haemaphysalis qinghaiensis Teng, 1980 (Acari:Ixodidae)

Haemaphysalis qinghaiensis is an endemic species and mainly inhabiting in the northwestern plateau of China, which can transmit many zoonotic pathogens and cause great harm to animals. In this study, the complete mitochondrial genome (mitogenome) of H. qinghaiensis was assembled through the Illumina HiSeq platform. The mitogenome was 14,533 bp in length, consisting of 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes and 3 noncoding regions (NCRs). The bias towards a high A+T content with 77.65% in mitogenome of H. qinghaiensis. The rearrangement of mitochondrial genes in H. qinghaiensis was consistent with other hard ticks. The phylogenetic analysis based on the concatenation of 13 PCGs from 65 tick mitogenomes showed that the H. qinghaiensis was clustered into a well-supported clade within the Haemaphysalis genus. This is the first complete mitogenome sequence of H. qinghaiensis, which provides a useful reference for understanding of the taxonomic and genetics of ticks.

Multiple bacterial partners in symbiosis with the nudibranch mollusk Rostanga alisae

The discovery of symbiotic associations extends our understanding of the biological diversity in the aquatic environment and their impact on the host’s ecology. Of particular interest are nudibranchs that unprotected by a shell and feed mainly on sponges. The symbiotic association of the nudibranch Rostanga alisae with bacteria was supported by ample evidence, including an analysis of cloned bacterial 16S rRNA genes and a fluorescent in situ hybridization analysis, and microscopic observations. A total of 74 clones belonging to the phyla α-, β-, γ-Proteobacteria, Actinobacteria, and Cyanobacteria were identified. FISH confirmed that bacteriocytes were packed with Bradyrhizobium, Maritalea, Labrenzia, Bulkholderia, Achromobacter, and Stenotrophomonas mainly in the foot and notum epidermis, and also an abundance of Synechococcus cyanobacteria in the intestinal epithelium. An ultrastructural analysis showed several bacterial morphotypes of bacteria in epidermal cells, intestine epithelium, and in mucus layer covering the mollusk body. The high proportion of typical bacterial fatty acids in R. alisae indicated that symbiotic bacteria make a substantial contribution to its nutrition. Thus, the nudibranch harbors a high diversity of specific endo- and extracellular bacteria, which previously unknown as symbionts of marine invertebrates that provide the mollusk with essential nutrients. They can provide chemical defense against predators.