Use of the 23S rRNA gene as a target template in the universal loop-mediated isothermal amplification (LAMP) of genomic DNA from phytoplasmas.

Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 'Candidatus Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of 'Candidatus Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.

Effects of ammonia nitrogen and organic carbon availability on microbial community structure and ecological interactions in a full-scale partial nitritation and anammox (PN/A) system.

Nutrient availability significantly impacts microbial biosynthesis, cell growth, and cell cycle progression. In this study, a full-scale plug-flow partial nitritation/anammox (PN/A) system was used to investigate variations in the microbial community structure in both immobilized carriers and flocs, as well as a gradual decrease in nutrient availability from upstream to downstream. We found that reduced ammonia nitrogen (from 150.4 to 30.6 mg/L) and organic carbon (from 415.7 to 342.8 mg/L) availability significantly lowered microbial diversity and altered microbial communities in biofilms other than flocs from upstream to downstream. The abundance of all anammox bacteria increased by 1.97 times, from 3.25 × 1010 to 6.40 × 1010 copies per gram of wet sludge, in the biofilm core microbiome. Furthermore, from upstream to downstream, taxa with lower ribosomal RNA operon copy numbers were consistently enriched in both biofilm and floc communities, indicating that slow-growing microorganisms are more likely to be enriched in low-nutrient environments. Rare taxa with a relative abundance of less than 0.1% exhibited unique metabolic functions, including amino acid, carbohydrate, cofactor, and vitamin metabolisms, which was inferred by PICRUST2 and persisted across the nutrient gradient in both the biofilm and floc communities. Despite their low abundance, they may play important roles in mediating the stability and function of the PN/A system. Overall, the results demonstrate the impact of a naturally formed ammonia nitrogen and organic carbon gradient in a full-scale plug-flow PN/A installation on nutrient availability and its effects on microbial diversity, community composition, and microbial interactions, which expands our fundamental understanding of this energy-efficient and promising biotechnology for treating high-strength ammonium wastewater.

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 (<500 bp) 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 ,000 entries, representing >10 ,000 prokaryotic 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 <3 s 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.

Three Ribosomal Operons of Escherichia coli Contain Genes Encoding Small RNAs That Interact With Hfq and CsrA in vitro.

Three out of the seven ribosomal RNA operons in Escherichia coli end in dual terminator structures. Between the two terminators of each operon is a short sequence that we report here to be an sRNA gene, transcribed as part of the ribosomal RNA primary transcript by read-through of the first terminator. The sRNA genes (rrA, rrB and rrF) from the three operons (rrnA, rrnB and rrnD) are more than 98% identical, and pull-down experiments show that their transcripts interact with Hfq and CsrA. Deletion of rrA, B, F, as well as overexpression of rrB, only modestly affect known CsrA-regulated phenotypes like biofilm formation, pgaA translation and glgC translation, and the role of the sRNAs in vivo may not yet be fully understood. Since RrA, B, F are short-lived and transcribed along with the ribosomal RNA components, their concentration reflect growth-rate regulation at the ribosomal RNA promoters and they could function to fine-tune other growth-phase-dependent processes in the cell. The primary and secondary structure of these small RNAs are conserved among species belonging to different genera of Enterobacteriales.

Genetic characterisation of Campylobacter concisus: Strategies for improved genomospecies discrimination.

Although at least two genetically distinct groups, or genomospecies, have been well documented for Campylobacter concisus, no phenotype has yet been identified for their differentiation and thus formal description as separate species. C. concisus has been isolated from a variety of sites in the human body, including saliva and stool samples from both healthy and diarrhoeic individuals. We evaluated the ability of a range of whole genome-based tools to distinguish between the two C. concisus genomospecies (GS) using a collection of 190 C. concisus genomes. Nine genomes from related Campylobacter species were included in some analyses to provide context. Analyses incorporating sequence analysis of multiple ribosomal genes generated similar levels of C. concisus GS discrimination as genome-wide comparisons. The C. concisus genomes formed two groups; GS1 represented by ATCC 33237T and GS2 by CCUG 19995. The two C. concisus GS were separated from the nine genomes of related species. GS1 and GS2 also differed in G+C content with medians of 37.56% and 39.51%, respectively. The groups are consistent with previously established GS and are supported by DNA reassociation results. Average Nucleotide Identity using MUMmer (ANIm) and Genome BLAST Distance Phylogeny generated in silico DNA-DNA hybridisation (isDDH) (against ATCC 33237T and CCUG 19995), plus G+C content provides cluster-independent GS discrimination suitable for routine use. Pan-genomic analysis identified genes specific to GS1 and GS2. WGS data and genomic species identification methods support the existence of two GS within C. concisus. These data provide genome-level metrics for strain identification to genomospecies level.

Is Oxford Nanopore sequencing ready for analyzing complex microbiomes?

This minireview will discuss the improvements in Oxford Nanopore (Oxford; sequencing technology that make the MinION a viable platform for microbial ecology studies. Specific issues being addressed are the increase in sequence accuracy from 65 to 96.5% during the last 5 years, the ability to obtain a quantifiable/predictive signal from the MinION with respect to target molecule abundance, simple-to-use GUI-based pathways for data analysis and the modest additional equipment needs for sequencing in the field. Coupling these recent improvements with the low capital costs for equipment and the reasonable per sample cost makes MinION sequencing an attractive option for virtually any laboratory.

Evaluating the Potential Antibiotic Resistance Status in Environment Based on the Trait of Microbial Community.

The overuse of antibiotics has promoted the propagation and dissemination of antibiotic resistance genes (ARGs) in environment. Due to the dense human population and intensive activities in coastal areas, the health risk of ARGs in coastal environment is becoming a severe problem. To date, there still lacks of a quantitative method to assess properly the gross antibiotic resistance at microbial community level. Here, we collected sediment samples from Hangzhou Bay (HB), Taizhou Bay (TB), and Xiangshan Bay (XB) of the East China Sea for community-level ARGs analysis. Based on the 16S rRNA genes and predictive metagenomics, we predicted the composition of intrinsic ARGs (piARGs) and some related functional groups. Firstly, a total of 40 piARG subtypes, belonging to nine drug classes and five resistance mechanisms, were obtained, among which the piARGs encoding multidrug efflux pumps were the most dominant in the three bays. Secondly, XB had higher relative abundances of piARGs and pathogens than the other two bays, which posed higher potential health risk and implied the heavier impact of long-term maricultural activities in this bay. Thirdly, the co-occurrence network analysis identified that there were more connections between piARGs and some potential pathogenic bacteria. Several piARG subtypes (e.g., tetA, aacA, aacC, and aadK) distributed widely in the microbial communities. And finally, the microbial diversity correlated negatively with the relative abundance of piARGs. Oil, salinity, and arsenic had significant effects on the variations of piARGs and potential pathogenic bacteria. The abundance-weighted average ribosomal RNA operon (rrn) copy number of microbial communities could be regarded as an indicator to evaluate the antibiotic resistance status. In conclusion, this study provides a new insight on how to evaluate antibiotic resistance status and their potential risk in environment based on a quantitative analysis of microbial communities.

High-Resolution Mapping of the Escherichia coli Chromosome Reveals Positions of High and Low Transcription.

Recent studies on targeted gene integrations in bacteria have demonstrated that chromosomal location can substantially affect a gene's expression level. However, these studies have only provided information on a small number of sites. To measure position effects on transcriptional propensity at high resolution across the genome, we built and analyzed a library of over 144,000 genome-integrated, standardized reporters in a single mixed population of Escherichia coli. We observed more than 20-fold variations in transcriptional propensity across the genome when the length of the chromosome was binned into broad 4 kbp regions; greater variability was observed over smaller regions. Our data reveal peaks of high transcriptional propensity centered on ribosomal RNA operons and core metabolic genes, while prophages and mobile genetic elements were enriched in less transcribable regions. In total, our work supports the hypothesis that E. coli has evolved gene-independent mechanisms for regulating expression from specific regions of its genome.

Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome.

rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.

Prevalence of Beauveria pseudobassiana among entomopathogenic fungi isolated from the hard tick, Ixodes ricinus.

Human and animal disease-transmitting hard ticks (Acari: Ixodidae) are of great concern for public health and animal farming. Alternatives to tick control by chemical acaricides are urgently needed, and one intensively evaluated biocontrol strategy is based on the use of tick-pathogenic filamentous fungi. An indispensable prerequisite for the development of tick-derived fungal isolates into registered myco-acaricides is their sound taxonomic characterisation. A set of fungal strains isolated from ixodid ticks in the Republic of Moldova was genetically characterised at the genus and species level together with further tick-derived fungal isolates from different geographic locations in Europe and North America. In a previous study, the same isolates had been assigned to the species Beauveria bassiana. Using a recent molecular taxonomic approach based on phylogenetic reconstruction from both internal transcribed spacer (ITS) and protein-encoding gene sequences, all fungi investigated were conclusively assigned to one of the two "hyphomycete" genera, Beauveria or Isaria (Ascomycota; Hypocreales; Cordycipitaceae). Within the genus Isaria, two species, Isaria farinosa and Isaria fumosorosea, were equally represented. Within the genus Beauveria, the comparatively rare species Beauveria pseudobassiana was found to strongly prevail among the isolates from Moldova, and one of the two tick-derived Beauveria strains from North America could be assigned to this species as well. In particular, the previous classification as B. bassiana could not be confirmed for any of the characterised tick pathogens from Europe and North America. The data presented here lend support to the hypothesis that within the genus Beauveria specific adaptation to ticks might have occurred within the species B. pseudobassiana. To test this hypothesis, a more extensive molecular taxonomic survey carefully reconsidering previous taxonomic assignments of tick-derived fungal isolates is needed.

Reprint of New opportunities for improved ribotyping of C. difficile clinical isolates by exploring their genomes.

Clostridium difficile causes outbreaks of infectious diarrhoea, most commonly occurring in healthcare institutions. Recently, concern has been raised with reports of C. difficile disease in those traditionally thought to be at low risk i.e. community acquired rather than healthcare acquired. This has increased awareness for the need to track outbreaks and PCR-ribotyping has found widespread use to elucidate epidemiologically linked isolates. PCR-ribotyping uses conserved regions of the 16S rRNA gene and 23S rRNA gene as primer binding sites to produce varying PCR products due to the intergenic spacer (ITS1) regions of the multiple operons. With the explosion of whole genome sequence data it became possible to analyse the start of the 23S rRNA gene for a more accurate selection of regions closer to the end of the ITS1. However the following questions must still be asked: (i) Does the chromosomal organisation of the rrn operon vary between C. difficile strains? and (ii) just how conserved are the primer binding regions? Eight published C. difficile genomes have been aligned to produce a detailed database of indels of the ITS1's from the rrn operon sets. An iPad Filemaker Go App has been constructed and named RiboTyping (RT). It contains detail such as sequences, ribotypes, strain numbers, GenBank numbers and genome position numbers. Access to various levels of the database is provided so that details can be printed. There are three main regions of the rrn operon that have been analysed by the database and related to each other by strain, ribotype and operon: (1) 16S gene (2) ITS1 indels (3) 23S gene. This has enabled direct intra- and inter-genomic comparisons at the strain, ribotype and operon (allele) levels in each of the three genomic regions. This is the first time that such an analysis has been done. By using the RT App with search criteria it will be possible to select probe combinations for specific strains/ribotypes/rrn operons for experiments to do with diagnostics, typing and recombination of operons. Many more incomplete C. difficile whole genome sequencing projects are recorded in GenBank as underway and the rrn operon information from these can also be added to the RT App when available. The RT App will help simplify probe selection because of the complexity of the ITS1 in C. difficile even in a single genome and because other allele-specific regions (16S and 23S genes) of variability can be relationally compared to design extra probes to increase sensitivity.