rRNA Operons Research Articles

Complete genome sequence of Promicromonospora sp. strain Populi, an actinobacterium isolated from Populus trichocarpa rhizosphere.

Promicromonospora sp. strain Populi is an actinobacterium isolated from the rhizosphere of a black cottonwood tree, Populus trichocarpa. We completely sequenced its 5.2-Mbp chromosome using Oxford Nanopore long reads and predicted it to encode 4,685 proteins, 3 rRNA operons, and 54 tRNAs and noncoding RNAs.

RRNA operon improves species-level classification of bacteria and microbial community analysis compared to 16S rRNA.

We quantitatively demonstrated that the rRNA operon outperformed the 16S rRNA and its V3-V4 regions in accuracy for both individual species identification and species-level microbial community analysis. Our findings can provide guidelines for selecting appropriate markers in the field of microbial research.

Microbial rrn copy number is associated with soil C: N ratio and pH under long-term fertilization

Soil microbial life-history strategies, as indicated by rRNA operon (rrn) copy numbers, strongly influence agro-ecosystem functioning. Long-term N fertilization causes strong and lasting changes in soil properties, yet its impact on microbial strategies remains largely unexplored. Using long-term field experiments across three agro-ecosystems, we consistently found that N fertilization strongly decreased soil C: N ratio and pH, further increasing the community-level rrn copy number, including both average rrn copy number and total 16S rRNA copy number. Soil C: N stoichiometry balanced by N supplement favored the growth of N-dependent copiotrophic species containing high rrn copy numbers (an average of 2.5) and increased their network connections, predominantly contributing to community-level rrn copy number increase. Decreased soil pH caused by N fertilization also favored the growth of some species whose abundances negatively correlated with pH, partially contributing to the community-level rrn copy number increase. By examining the genomes of two dominant species, we found that microorganisms with a higher rrn copy number (6), e.g., Streptomyces scabiei, possessed more genes related to C and N transport and metabolism. In contrast, the Mycobacterium simiae with a lower rrn copy number (1) has more genes associated with secondary metabolite biosynthesis and lipid transport and metabolism. Our finding challenges the concept of microbial life-strategy regulation solely by nutrient availability, highlighting the important contributions of soil stoichiometric balance and pH to microbial strategies in agro-ecosystems under long-term N inputs.

Acidisoma cladoniae sp. nov., an acidotolerant bacterium isolated from an Antarctic lichen.

Gram-staining-negative, aerobic, white-cream-pearly colony, coccobacilli, and non-motile bacterial strain, PAMC 29798T was isolated from an Antarctic lichen. The strain was acidotolerant and psychrotolerant growing at pH 4.0-7.5 (optimally at pH 4.0-6.5) and 0-25°C (optimally at 10-20°C). The major fatty acids are Summed Feature 8, C18:1 2OH, and C19:0 cyclo ω8c. The major respiratory quinone was Q-10. Phylogenetic and phylogenomic analyses indicated that strain PAMC 29798T belonged to the genus Acidisoma and 16S rRNA gene sequences of PAMC 29798T were closely related to Acidisoma silvae (97.7% sequence similarity), Acidisoma cellulosilyticum (96.5%), Acidisoma tundrae (96.5%), and Acidisoma sibiricum (96.3%). Genomic relatedness analyses showed that strain PAMC 29798T was clearly distinguished from type strains of the genus Acidisoma based on values of average nucleotide identity (< 75%) and the digital DNA-DNA hybridization (< 19.6%). Genome analysis revealed that the genome size of PAMC 29798T is approximately 5.0 Mb with a G+C content of 63.4%. The complete genome comprises 5 contigs containing 4636 protein-coding genes, 46 tRNA genes, and 2 rRNA operons. The genome possesses genes for light-harvesting complexes, type-II photosynthetic reaction center, and C-P lyase to solubilize organic phosphates, while genes encoding nitrogenase iron protein involved in the nitrogen fixation were not present. Based on the results of phylogenetic, genome-based relatedness, and physiological and genomic analyses, strain PAMC 29798T is proposed to represent a novel species of the genus Acidisoma, with the name Acidisoma cladoniae. The type strain is PAMC 29798T (= KCTC 82159T = JCM 35634T).

Clustering of rRNA operons in E. coli is disrupted by σH.

Chromosomal organization in E. coli as examined by Hi-C methodology indicates that long-range interactions are sparse. Yet, spatial co-localization or 'clustering' of 6/7 ribosomal RNA (rrn) operons distributed over half the 4.6 Mbp genome has been captured by two other methodologies - fluorescence microscopy and Mu transposition. Our current understanding of the mechanism of clustering is limited to mapping essential cis elements. To identify trans elements, we resorted to perturbing the system by chemical and physical means and observed that heat shock disrupts clustering. Levels of σH are known to rise as a cellular response to the shock. We show that elevated expression of σH alone is sufficient to disrupt clustering, independent of heat stress. The anti-clustering activity of σH does not depend on its transcriptional activity but requires core-RNAP interaction and DNA-binding activities. This activity of σH is suppressed by ectopic expression of σD suggesting a competition for core-RNAP. A query of the other five known σ factors of E. coli found that elevated expression of FecI, the ECF σ factor that controls iron citrate transport, also perturbs clustering and is also suppressed by σD. We discuss a possible scenario for how these membrane-associated σ factors participate in clustering of distant rrn loci.

Temporal changes in the mouse gut bacteriota influenced by host sex, diet, and exercise.

The gut microbiota plays an important role in host physiology. However, the effects of host sex, lifestyle, and temporal influences on the bacterial community within the gut remain ill-defined. To address this gap, we evaluated 56 male and female mice over a 10-wk study to assess the effects of sex, diet, and exercise on gut community dynamics. Mice were randomly assigned to high-fat or control diet feeding and had free access to running wheels or remained sedentary throughout the study period. The fecal bacterial community was characterized by rRNA operon profiling via nanopore sequencing. Differential abundance testing indicated that ∼200 bacterial taxa were significantly influenced by sex, diet, or exercise (4.2% of total community), which also changed over time (82 taxa, 1.7% of total community). Phylogenetic analysis of taxa closely related to Dysosmobacter welbionis, and several members of the family Muribaculaceae were examined more closely and demonstrated distinct species/strain level subclustering by host sex, diet, and exercise. Collectively, these data suggest that sex and lifestyle can alter the gut bacteriota at the species/strain level that may play a role in host health. These results also highlight the need for improved characterization methods to survey microbial communities at finer taxonomic resolution.NEW & NOTEWORTHY This study demonstrates that host sex, diet, and exercise can each modulate gut bacterial community structure, which may have consequences to host physiology. Our analysis shows selection of novel strains and genera for some members of the Oscillospiraceae and Muribaculaceae by host sex, diet, and activity status. Overall, these findings provide a framework for detecting the next generation of beneficial bacteria targeting obesity and associated metabolic diseases in a sex-specific manner.

Plant growth-promoting microorganisms drive K strategists through deterministic processes to alleviate biological stress caused by Fusarium oxysporum

Soybean root rot, caused by soil-borne pathogens such as Fusarium oxysporum, frequently occurs in Northeast China and leads to a decline in soil health and becoming a bottleneck for soybean yield in the region. To address this issue, applying beneficial microorganisms and altering soil microbial community structure have become effective strategies. In this study, the 90-day soybean pot experiment was conducted to explore the assembly process and life strategy selection of bacterial communities in the rhizosphere of healthy (inoculated with Funneliformis mosseae, F group and treated with Pseudomonas putida, P group) and diseased (inoculated with F. oxysporum, O group) soybean plants, as well as the recovery effect of beneficial microorganisms on soil-borne diseases (combined treatments OP and OF). Results indicated that in healthy soils (P and F), microbial community assembly process in the soybean rhizosphere was entirely governed by heterogeneous selection (HeS, 100 %). However, inoculated with P. putida (OP) was primarily driven by stochastic processes (HeS 40 %, dispersal limitation (DL) 60 %), and the F. mosseae treatment (OF) predominantly followed a deterministic process (HeS 89 %, DL 11 %) in diseased soils. Inoculation of plant growth-promoting microorganisms (PGPMs) in diseased soil drove the life strategy of the rhizosphere bacterial community from r- to K-strategy, evident from the lower rRNA operon (rrn) copy numbers (O 3.7, OP 2.1, OF 2.3), higher G+ to G- ratios (O 0.47, OP 0.58, OF 0.57), and a higher abundance of oligotrophs (O 50 %, OP 53 %, OF 54 %). In healthy (P and F) and diseased (O, OP, OF) rhizosphere soils, OTU820, OTU6142, and OTU8841 under the K-strategy, and OTU6032 and OTU6917 under the r-strategy, which served as keystone species, had a significant promoting relationship with plant biomass and defense capabilities ( p ＜0.05). Additionally, inoculation of PGPMs improved autotoxin degradation and positively correlated with bacterial life strategies in both healthy and diseased soils (P, F, OP and OF) ( p ＜0.05). These findings enhance our understanding of soil-microbe interactions and offer new insights and precise control measures for soybean disease management and soil environment remediation.

Genetic basis of macrolide resistance in porcine Pasteurella multocida isolates from the German national resistance monitoring program GERM-Vet 2008-2021.

To analyse porcine Pasteurella multocida isolates obtained from the national resistance monitoring program GERM-Vet 2008-2021 in Germany for phenotypic and genotypic macrolide resistance. The antimicrobial susceptibility of the isolates was determined by broth microdilution according to CLSI standards. Closed genomes were obtained by WGS via Illumina MiSeq and MinION platforms followed by a hybrid assembly. Of 1114 porcine P. multocida isolates, only four isolates (0.36%), one each from 2008, 2010, 2019 and 2021, exhibited resistance to at least one macrolide tested. The isolate from 2010 was only resistant to erythromycin and WGS analysis neither revealed a macrolide resistance gene nor a macrolide resistance-mediating mutation. The isolates from 2008 and 2019 were resistant to erythromycin, tilmicosin, tildipirosin, tulathromycin and gamithromycin and showed either only the A2058G mutation in all six 23S rRNA operons or the chromosomally located macrolide resistance genes msr(E) and mph(E), respectively. The isolate from 2021 was resistant to erythromycin, tulathromycin, gamithromycin and tylosin and carried a novel integrative and conjugative element of 64 966 bp, designated Tn7730, in its chromosomal DNA. It harboured the macrolide resistance genes mef(C), mph(G) and estT, the lincosamide resistance gene lnu(H), and the tetracycline resistance gene tet(Y), the last two were detected for the first time in P. multocida. Macrolide resistance in German porcine P. multocida can be due to resistance-mediating mutations or resistance genes. The presence of the novel Tn7730 carrying three different macrolide resistance genes is alarming and should be monitored.

Antarctic Soils Select Copiotroph-Dominated Bacteria.

The life strategies of bacterial communities determine their structure and function and are an important driver of biogeochemical cycling. However, the variations in these strategies under different soil resource conditions remain largely unknown. We explored the bacterial life strategies and changes in structure and functions between Antarctic soils and forest (temperate, subtropical, and tropical) soils. The results showed that the weighted mean rRNA operon copy number in temperate soils was 19.5% lower than that in Antarctic soils, whereas no significant differences were observed among Antarctic, subtropical, and tropical soils. An unexpected result was that bacterial communities in Antarctic soils tended to be copiotrophs, such as Actinobacteriota and Bacteroidota, whereas those in temperate soils tended to be oligotrophs, such as Acidobacteriota and Chloroflexi. Functional predictions showed that in comparison to copiotrophs in Antarctic soils, temperate-inhabiting oligotrophic bacteria exhibited an 84.2-91.1% lower abundance of labile C decomposition genes (hemicellulose, cellulose, monosaccharides, and disaccharides), whereas a 74.4% higher abundance of stable C decomposition (lignin). Genes involved in N cycling (nitrogen fixation, assimilatory nitrate reduction, and denitrification) were 24.3-64.4% lower in temperate soils than in Antarctic soils. Collectively, our study provides a framework for describing the life strategies of soil bacteria, which are crucial to global biogeochemical cycles.

Genotypic and Phenotypic Characterization of Pseudomonas atacamensis EMP42 a PGPR Strain Obtained from the Rhizosphere of Echinocactus platyacanthus (Sweet Barrel).

Plant growth-promoting rhizobacteria (PGPR) are a group of bacteria that associate with the rhizosphere of plants; one of the most abundant bacterial genera in this ecological niche is Pseudomonas, which is constantly expanding due to the emergence of new species such as Pseudomonas atacamensis, whose discovery in 2019 has led to the characterization of several strains from different environments but taxonomically related. The objective of this work was to phenotypically and molecularly characterize P. atacamensis strain EMP42, isolated from the rhizosphere of Echinocactus platyacanthus. The strain EMP42 is able to use different substrates and reduce oxidative stress in plants. It is capable of improving growth parameters such as the number of inflorescences and the height of the aerial body of Arabidopsis thaliana, as well as the germination and seedling survival of the cacti Echinocactus platyacanthus and Astrophytum capricorne. The genetic structure of P. atacamensis EMP42 consists of a closed chromosome of 6.14 Mbp, and 61.1% GC content. It has 5572 genes, including those associated with PGPR activities, such as the trpABCDE, SAP, phoABPRU and acsABC genes, among others, and three ncRNA loci, nine regulatory regions, five complete rRNA operons and three CRISPR-Cas loci, showing phylogenomic similarities with the reference strain P. atacamensis B21-026. Therefore, this study contributes to the understanding of genomic diversity within P. atacamensis and, particularly, highlights the potential application of strain EMP42 as a PGPR.

Fine-scale characterization of the soybean rhizosphere microbiome via synthetic long reads and avidity sequencing

BackgroundThe rhizosphere microbiome displays structural and functional dynamism driven by plant, microbial, and environmental factors. While such plasticity is a well-evidenced determinant of host health, individual and community-level microbial activity within the rhizosphere remain poorly understood, due in part to the insufficient taxonomic resolution achieved through traditional marker gene amplicon sequencing. This limitation necessitates more advanced approaches (e.g., long-read sequencing) to derive ecological inferences with practical application. To this end, the present study coupled synthetic long-read technology with avidity sequencing to investigate eukaryotic and prokaryotic microbiome dynamics within the soybean (Glycine max) rhizosphere under field conditions.ResultsSynthetic long-read sequencing permitted de novo reconstruction of the entire 18S-ITS1-ITS2 region of the eukaryotic rRNA operon as well as all nine hypervariable regions of the 16S rRNA gene. All full-length, mapped eukaryotic amplicon sequence variants displayed genus-level classification, and 44.77% achieved species-level classification. The resultant eukaryotic microbiome encompassed five kingdoms (19 genera) of protists in addition to fungi – a depth unattainable with conventional short-read methods. In the prokaryotic fraction, every full-length, mapped amplicon sequence variant was resolved at the species level, and 23.13% at the strain level. Thirteen species of Bradyrhizobium were thereby distinguished in the prokaryotic microbiome, with strain-level identification of the two Bradyrhizobium species most reported to nodulate soybean. Moreover, the applied methodology delineated structural and compositional dynamism in response to experimental parameters (i.e., growth stage, cultivar, and biostimulant application), unveiled a saprotroph-rich core microbiome, provided empirical evidence for host selection of mutualistic taxa, and identified key microbial co-occurrence network members likely associated with edaphic and agronomic properties.ConclusionsThis study is the first to combine synthetic long-read technology and avidity sequencing to profile both eukaryotic and prokaryotic fractions of a plant-associated microbiome. Findings herein provide an unparalleled taxonomic resolution of the soybean rhizosphere microbiota and represent significant biological and technological advancements in crop microbiome research.

Benchmarking long-read sequencing strategies for obtaining ASV-resolved rRNA operons from environmental microeukaryotes.

The use of short-read metabarcoding for classifying microeukaryotes is challenged by the lack of comprehensive 18S rRNA reference databases. While recent advances in high-throughput long-read sequencing provide the potential to greatly increase the phylogenetic coverage of these databases, the performance of different sequencing technologies and subsequent bioinformatics processing remain to be evaluated, primarily because of the absence of well-defined eukaryotic mock communities. To address this challenge, we created a eukaryotic rRNA operon clone-library and turned it into a precisely defined synthetic eukaryotic mock community. This mock community was then used to evaluate the performance of three long-read sequencing strategies (PacBio circular consensus sequencing and two Nanopore approaches using unique molecular identifiers) and three tools for resolving amplicons sequence variants (ASVs) (USEARCH, VSEARCH, and DADA2). We investigated the sensitivity of the sequencing techniques based on the number of detected mock taxa, and the accuracy of the different ASV-calling tools with a specific focus on the presence of chimera among the final rRNA operon ASVs. Based on our findings, we provide recommendations and best practice protocols for how to cost-effectively obtain essentially error-free rRNA operons in high-throughput. An agricultural soil sample was used to demonstrate that the sequencing and bioinformatic results from the mock community also translates to highly diverse natural samples, which enables us to identify previously undescribed microeukaryotic lineages.

Enhancing soil petrochemical contaminant remediation through nutrient addition and exogenous bacterial introduction

Biostimulation (providing favorable environmental conditions for microbial growth) and bioaugmentation (introducing exogenous microorganisms) are effective approaches in the bioremediation of petroleum-contaminated soil. However, uncertainty remains in the effectiveness of these two approaches in practical application. In this study, we constructed mesocosms using petroleum hydrocarbon-contaminated soil. We compared the effects of adding nutrients, introducing exogenous bacterial degraders, and their combination on remediating petroleum contamination in the soil. Adding nutrients more effectively accelerated total petroleum hydrocarbon (TPH) degradation than other treatments in the initial 60 days’ incubation. Despite both approaches stimulating bacterial richness, the community turnover caused by nutrient addition was gentler than bacterial degrader introduction. As TPH concentrations decreased, we observed a succession in microbial communities characterized by a decline in copiotrophic, fast-growing bacterial r-strategists with high rRNA operon (rrn) copy numbers. Ecological network analysis indicated that both nutrient addition and bacterial degrader introduction enhanced the complexity and stability of bacterial networks. Compared to the other treatment, the bacterial network with nutrient addition had more keystone species and a higher proportion of negative associations, factors that may enhance microbial community stability. Our study demonstrated that nutrient addition effectively regulates community succession and ecological interaction to accelerate the soil TPH degradation.

Minimal impact of nitrogen addition on bacterial and fungal communities during fungal necromass decomposition in a subalpine coniferous plantation

Fungal necromass is a vital source of stable carbon and available nitrogen in soils, especially in coniferous forests that are extensively colonized by ectomycorrhizal fungi. Here, we examined changes in the necromass microbial community during a 20-week incubation experiment in a subalpine coniferous plantation. We also explored how the necromass microbial community responded to simulated nitrogen deposition, a global change factor that substantially affects nitrogen-limited coniferous forests. The results showed that necromass microbial community was more dominated by copiotrophic bacteria with high community average rRNA operon copy number (3.1–6.4) and fast-growing fungi (moulds and yeasts) than surrounding soils. Ectomycorrhizal fungi also constituted a significant portion of the fungal community during the fast decay stage (first 5 weeks) of necromass decomposition, indicating that fungal necromass may serve as a nitrogen source for the nitrogen-limited trees. The shifts in microbial functional compositions (e.g., decreased proportions of copiotrophic bacteria) as decomposition progressed were partly due to alterations in substrate chemistry (i.e., the levels of aliphatic compounds including soluble carbohydrate, amorphous glucan polymers, and crystalline glucans and chitin). In contrast, nitrogen addition had limited influences on microbial diversity and composition, likely due to the nitrogen-rich nature of the fungal necromass. Overall, our findings reinforce the existence of a specific and dynamic core necrobiome in fungal necromass and enhance our understanding of how fungal necrobiome responds to environmental changes.

GROND: a quality-checked and publicly available database of full-length 16S-ITS-23S rRNA operon sequences.

Sequence comparison of 16S rRNA PCR amplicons is an established approach to taxonomically identify bacterial isolates and profile complex microbial communities. One potential application of recent advances in long-read sequencing technologies is to sequence entire rRNA operons and capture significantly more phylogenetic information compared to sequencing of the 16S rRNA (or regions thereof) alone, with the potential to increase the proportion of amplicons that can be reliably classified to lower taxonomic ranks. Here we describe GROND (Genome-derived Ribosomal Operon Database), a publicly available database of quality-checked 16S-ITS-23S rRNA operons, accompanied by multiple taxonomic classifications. GROND will aid researchers in analysis of their data and act as a standardised database to allow comparison of results between studies.

Whole-genome sequence of the strictly anaerobic bacterial strain SANA belonging to the family Gottschalkiaceae, isolated from a xenic culture of an anaerobic protist.

An anaerobic bacterial strain SANA was isolated from a xenic culture of an anaerobic heterolobosean protist which was obtained from a saline lake in Japan. Its draft genome comprises 1 circular chromosome (3,490,293 bp), harboring 3,275 predicted protein-coding and 73 tRNA-encoding genes and 8 rRNA operons.

Development of Highly Efficient Universal Pneumocystis Primers and Their Application in Investigating the Prevalence and Genetic Diversity of Pneumocystis in Wild Hares and Rabbits.

Despite its ubiquitous infectivity to mammals with strong host specificity, our current knowledge about Pneumocystis has originated from studies of merely 4% of extant mammalian species. Further studies of Pneumocystis epidemiology across a broader range of animal species require the use of assays with high sensitivity and specificity. To this end, we have developed multiple universal Pneumocystis primers targeting different genetic loci with high amplification efficiency. Application of these primers to PCR investigation of Pneumocystis in free-living hares (Lepus townsendii, n = 130) and rabbits (Oryctolagus cuniculus, n = 8) in Canada revealed a prevalence of 81% (105/130) and 25% (2/8), respectively. Genotyping analysis identified five and two variants of Pneumocystis from hares and rabbits, respectively, with significant sequence divergence between the variants from hares. Based on phylogenetic analysis using nearly full-length sequences of the mitochondrial genome, nuclear rRNA operon and dihydropteroate synthase gene for the two most common variants, Pneumocystis in hares and rabbits are more closely related to each other than either are to Pneumocystis in other mammals. Furthermore, Pneumocystis in both hares and rabbits are more closely related to Pneumocystis in primates and dogs than to Pneumocystis in rodents. The high prevalence of Pneumocystis in hares (P. sp. 'townsendii') suggests its widespread transmissibility in the natural environment, similar to P. oryctolagi in rabbits. The presence of multiple distinct Pneumocystis populations in hares contrasts with the lack of apparent intra-species heterogeneity in P. oryctolagi, implying a unique evolution history of P. sp. 'townsendii' in hares.

Whole-genome sequence of a marine bacterial strain, FRT2, belonging to the genus Leeuwenhoekiella, isolated from the seawater of the Obama Bay in Fukui, Japan.

A marine bacterial strain, FRT2, was isolated from the surface water of the Obama Bay in Fukui, Japan. Its complete genome comprises one circular chromosome (3,806,097 bp), harboring 3,269 predicted protein- and 44-tRNA-encoding genes and 3 rRNA operons.

Harnessing the intragenomic variability of rRNA operons to improve differentiation of Vibrio species

Although the 16S rRNA gene is frequently used as a phylogenetic marker in analysis of environmental DNA, this marker often fails to distinguish closely related species, including those in the genus Vibrio. Here, we investigate whether inclusion and analysis of 23S rRNA sequence can help overcome the intrinsic weaknesses of 16S rRNA analyses for the differentiation of Vibrio species. We construct a maximum likelihood 16S rRNA gene tree to assess the use of this gene to identify clades of Vibrio species. Within the 16S rRNA tree, we identify the putative informative bases responsible for polyphyly, and demonstrate the association of these positions with tree topology. We demonstrate that concatenation of 16S and 23S rRNA genes increases the number of informative nucleotide positions, thereby overcoming ambiguities in 16S rRNA-based phylogenetic reconstructions. Finally, we experimentally demonstrate that this approach considerably improves the differentiation and identification of Vibrio species in environmental samples.

Draft genome sequence of the deep-subsurface Ciceribacter sp. strain T2.26MG-112.2, a second Rhizobiaceae isolated from the Iberian Pyrite Belt at 492.6 mbs.

T2.26MG-112.2 is a Ciceribacter strain that has been isolated from the deep subsurface of the Iberian Pyrite Belt. We report its draft genome consisting of a chromosome of ≈4.9 Mb and a plasmid of 357 kb. The annotation reveals 4,824 coding sequences, 48 tRNA genes, and 1 rRNA operon.