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.

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.

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.

Fibrolytic rumen bacteria of camel and sheep and their applications in the bioconversion of barley straw to soluble sugars for biofuel production

Lignocellulosic biomass such as barley straw is a renewable and sustainable alternative to traditional feeds and could be used as bioenergy sources; however, low hydrolysis rate reduces the fermentation efficiency. Understanding the degradation and colonization of barley straw by rumen bacteria is the key step to improve the utilization of barley straw in animal feeding or biofuel production. This study evaluated the hydrolysis of barley straw as a result of the inoculation by rumen fluid of camel and sheep. Ground barley straw was incubated anaerobically with rumen inocula from three fistulated camels (FC) and three fistulated sheep (FR) for a period of 72 h. The source of rumen inoculum did not affect the disappearance of dry matter (DMD), neutral detergent fiber (NDFD). Group FR showed higher production of glucose, xylose, and gas; while higher ethanol production was associated with cellulosic hydrolysates obtained from FC group. The diversity and structure of bacterial communities attached to barley straw was investigated by Illumina Mi-Seq sequencing of V4-V5 region of 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes and Bacteroidetes. The dominant genera were RC9_gut_group, Ruminococcus, Saccharofermentans, Butyrivibrio, Succiniclasticum, Selenomonas, and Streptococcus, indicating the important role of these genera in lignocellulose fermentation in the rumen. Group FR showed higher RC9_gut_group and group FC revealed higher Ruminococcus, Saccharofermentans, and Butyrivibrio. Higher enzymes activities (cellulase and xylanase) were associated with group FC. Thus, bacterial communities in camel and sheep have a great potential to improve the utilization lignocellulosic material in animal feeding and the production of biofuel and enzymes.

Robust expansion of phylogeny for fast-growing genome sequence data

Massive sequencing of SARS-CoV-2 genomes has led to a great demand for adding new samples to a reference phylogeny instead of building the tree from scratch. To address such challenge, we proposed an algorithm 'TIPars' by integrating parsimony analysis with pre-computed ancestral sequences. Compared to four state-of-the-art methods on four benchmark datasets (SARS-CoV-2, Influenza virus, Newcastle disease virus and 16S rRNA genes), TIPars achieved the best performance in most tests. It took only 21 seconds to insert 100 SARS-CoV-2 genomes to a 100k-taxa reference tree using near 1.4 gigabytes of memory. Its efficient and accurate phylogenetic placements and incrementation for phylogenies with highly similar and divergent sequences suggest that it will be useful in a wide range of studies including pathogen molecular epidemiology, microbiome diversity and systematics.

Taxonomic composition and biodiversity of the gut microbiome from patients with irritable bowel syndrome, ulcerative colitis, and asthma

To date, the association of an imbalance of the intestinal microbiota with various human diseases, including both diseases of the gastrointestinal tract and disorders of the immune system, has been shown. However, despite the huge amount of accumulated data, many key questions still remain unanswered. Given limited data on the composition of the gut microbiota in patients with ulcerative colitis (UC) and irritable bowel syndrome (IBS) from different parts of Siberia, as well as the lack of data on the gut microbiota of patients with bronchial asthma (BA), the aim of the study was to assess the biodiversity of the gut microbiota of patients with IBS, UC and BA in comparison with those of healthy volunteers (HV). In this study, a comparative assessment of the biodiversity and taxonomic structure of gut microbiome was conducted based on the sequencing of 16S rRNA genes obtained from fecal samples of patients with IBS, UC, BA and volunteers. Sequences of the Firmicutes and Bacteroidetes types dominated in all samples studied. The third most common in all samples were sequences of the Proteobacteria type, which contains pathogenic and opportunistic bacteria. Sequences of the Actinobacteria type were, on average, the fourth most common. The results showed the presence of dysbiosis in the samples from patients compared to the sample from HVs. The ratio of Firmicutes/Bacteroidetes was lower in the IBS and UC samples than in HV and higher the BA samples. In the samples from patients with intestinal diseases (IBS and UC), an increase in the proportion of sequences of the Bacteroidetes type and a decrease in the proportion of sequences of the Clostridia class, as well as the Ruminococcaceae, but not Erysipelotrichaceae family, were found. The IBS, UC, and BA samples had signif icantly more Proteobacteria sequences, including Methylobacterium, Sphingomonas, Parasutterella, Halomonas, Vibrio, as well as Escherichia spp. and Shigella spp. In the gut microbiota of adults with BA, a decrease in the proportion of Roseburia, Lachnospira, Veillonella sequences was detected, but the share of Faecalibacterium and Lactobacillus sequences was the same as in healthy individuals. A signif icant increase in the proportion of Halomonas and Vibrio sequences in the gut microbiota in patients with BA has been described for the f irst time.

Investigation of Sources, Diversity, and Variability of Bacterial Aerosols in Athens, Greece: A Pilot Study

We characterized the composition, diversity, and potential bacterial aerosol sources in Athens’ urban air by DNA barcoding (analysis of 16S rRNA genes) during three seasons in 2019. Air samples were collected using the recently developed Rutgers Electrostatic Passive Sampler (REPS). It is the first field application of REPS to study bacterial aerosol diversity. REPS samplers captured a sufficient amount of biological material to demonstrate the diversity of airborne bacteria and their variability over time. Overall, in the air of Athens, we detected 793 operational taxonomic units (OTUs), which were fully classified into the six distinct taxonomic categories (Phylum, Class, Order, etc.). These OTUs belonged to Phyla Actinobacteria, Firmicutes, Proteobacteria, Bacteroidetes, Cyanobacteria, and Fusobacteria. We found a complex community of bacterial aerosols with several opportunistic or potential pathogens in Athens’ urban air. Referring to the available literature, we discuss the likely sources of observed airborne bacteria, including soil, plants, animals, and humans. Our results on bacterial diversity are comparable to earlier studies, even though the sampling sites are different or geographically distant. However, the exact functional and ecological role of bioaerosols and, even more importantly, their impact on public health and the ecosystem requires further air monitoring and analysis.

Detection, in silico analysis and molecular diversity of phytoplasmas from solanaceous crops in Turkey

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

Isolating and Characterizing Phosphorus Solubilizing Bacteria From Rhizospheres of Native Plants Grown in Calcareous Soils

Many soils including urban soils have high legacy soil phosphorus (P) due to repeated applications of P fertilizers, but a large portion legacy soil P is fixed by calcium in the calcareous soils. Phosphorus solubilizing bacteria (PSB) have the ability to transfer fixed (non-labile) soil P into bioavailable P. The aim of this study was to isolate P solubilizing bacteria from the rhizospheres of four local native plants [broomsedge bluestem (Andropogon virginicus), giant sword ferns (Nephrolepis biserrata), sawgrass (Cladium jamaicense), and sea ox-eye daisy (Borrichia frutescens)] grown in low bioavailable P calcareous soils and to determine their ability to solubilize P. A total of 44 strains of PSB were isolated with 15 of them being identified by sequencing the 16S rRNA genes as Bacillus flexus, Beijerinckia fluminensis, Enterobacter ludwigii, Enterobacter sp., and Pantoea cypripedii. After a 7-day incubation, these strains reduced pH to &lt;4.27 and increased water-soluble P up to 588 mg L−1. Enterobacter ludwigii showed superior P solubilizing ability amount PSB isolated. Therefore, the isolated strains from the local native environment have the potential to thrive in local calcareous soils and possess strong ability to transform non-labile P into bioavailable forms for plants to uptake.