Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination.

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction-modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.

P finder: genomic and metagenomic annotation of RNase P RNA gene (rnpB).

BACKGROUND: The rnpB gene encodes for an essential catalytic RNA (RNase P). Like other essential RNAs, RNase P's sequence is highly variable. However, unlike other essential RNAs (i.e. tRNA, 16 S, 6 S,...) its structure is also variable with at least 5 distinct structure types observed in prokaryotes. This structural variability makes it labor intensive and challenging to create and maintain covariance models for the detection of RNase P RNA in genomic and metagenomic sequences. The lack of a facile and rapid annotation algorithm has led to the rnpB gene being the most grossly under annotated essential gene in completed prokaryotic genomes with only a 24% annotation rate. Here we describe the coupling of the largest RNase P RNA database with the local alignment scoring algorithm to create the most sensitive and rapid prokaryote rnpB gene identification and annotation algorithm to date. RESULTS: Of the 2772 completed microbial genomes downloaded from GenBank only 665 genomes had an annotated rnpB gene. We applied P Finder to these genomes and were able to identify 2733 or nearly 99% of the 2772 microbial genomes examined. From these results four new rnpB genes that encode the minimal T-type P RNase P RNAs were identified computationally for the first time. In addition, only the second C-type RNase P RNA was identified in Sphaerobacter thermophilus. Of special note, no RNase P RNAs were detected in several obligate endosymbionts of sap sucking insects suggesting a novel evolutionary adaptation. CONCLUSIONS: The coupling of the largest RNase P RNA database and associated structure class identification with the P Finder algorithm is both sensitive and rapid, yielding high quality results to aid researchers annotating either genomic or metagenomic data. It is the only algorithm to date that can identify challenging RNAse P classes such as C-type and the minimal T-type RNase P RNAs. P Finder is written in C# and has a user-friendly GUI that can run on multiple 64-bit windows platforms (Windows Vista/7/8/10). P Finder is free available for download at https://github.com/JChristopherEllis/P-Finder as well as a small sample RNase P RNA file for testing.

Kistimonas alittae sp. nov., a gammaproteobacterium isolated from the marine annelid Alitta succinea.

A novel Gram-stain-negative, rod-shaped, motile, non-spore-forming, facultatively anaerobic marine bacterium was isolated from the gastrointestinal tract of the sandworm Alitta succinea collected from Grice Cove, South Carolina, USA. The strain was arginine dihydrolase-positive, and oxidase- and catalase-positive. Growth occurred between 10 and 37 °C, with optimal growth occurring between 30 and 32 °C. Comparative 16S rRNA gene sequence analysis showed its nearest neighbours are members of the genus Kistimonas of the family Hahellaceae, which is found in the order Oceanospirillales, class Gammaproteobacteria. The closest related species was Kistimonas asteriae KMD 001T with 16S rRNA gene sequence similarity of 99.0 %. However, DNA-DNA hybridization between these strains revealed less than 70 % DNA-DNA relatedness, supporting the novel species status of the strain. The major fatty acids were C16 : 0, C18 : 0, C18 : 1ω7c and a summed feature that contained C16 : 1ω6c/C16 : 1ω7c. The major respiratory quinone was ubiquinone-9 and the predominant polar lipids were phosphatidylserine, phosphoethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content was 52.5 mol%. Based on the data presented, strain BGP-2T is considered to represent a novel member of the genus Kistimonas, for which the name Kistimonas alittae sp. nov. is proposed. The type strain is BGP-2T (=CCUG 65711T=JCM 30010T).

Fungal Community Responses to Past and Future Atmospheric CO2 Differ by Soil Type.

Soils sequester and release substantial atmospheric carbon, but the contribution of fungal communities to soil carbon balance under rising CO2 is not well understood. Soil properties likely mediate these fungal responses but are rarely explored in CO2 experiments. We studied soil fungal communities in a grassland ecosystem exposed to a preindustrial-to-future CO2 gradient (250 to 500 ppm) in a black clay soil and a sandy loam soil. Sanger sequencing and pyrosequencing of the rRNA gene cluster revealed that fungal community composition and its response to CO2 differed significantly between soils. Fungal species richness and relative abundance of Chytridiomycota (chytrids) increased linearly with CO2 in the black clay (P < 0.04, R(2) > 0.7), whereas the relative abundance of Glomeromycota (arbuscular mycorrhizal fungi) increased linearly with elevated CO2 in the sandy loam (P = 0.02, R(2) = 0.63). Across both soils, decomposition rate was positively correlated with chytrid relative abundance (r = 0.57) and, in the black clay soil, fungal species richness. Decomposition rate was more strongly correlated with microbial biomass (r = 0.88) than with fungal variables. Increased labile carbon availability with elevated CO2 may explain the greater fungal species richness and Chytridiomycota abundance in the black clay soil, whereas increased phosphorus limitation may explain the increase in Glomeromycota at elevated CO2 in the sandy loam. Our results demonstrate that soil type plays a key role in soil fungal responses to rising atmospheric CO2.

The small nucleolar ribonucleoprotein (snoRNP) database.

Small nucleolar ribonucleoproteins (snoRNPs) are widely studied and characterized as guide RNAs for sequence-specific 2'-O-ribose methylation and psuedouridylation of ribosomal RNAs. In addition, snoRNAs have also been shown to interact with some tRNAs and direct alternative splicing in mRNA biogenesis. Recent advances in bioinformatics have resulted in new algorithms able to rapidly identify noncoding RNAs generally and snoRNAs specifically in genomic and metagenomic sequences, resulting in a rapid increase in the number and diversity of identified snoRNA sequences. The snoRNP database is a web-based collection of snoRNA and snoRNA-associated protein sequences from a wide range of species. The database currently contains 8994 snoRNA sequences from Bacteria, Archaea, and Eukaryotes and 589 snoRNA-associated protein sequences. The snoRNP database can be found at: http://evolveathome.com/snoRNA/snoRNA.php.

Bioprospecting Trichoderma: A Systematic Roadmap to Screen Genomes and Natural Products for Biocontrol Applications.

Natural products derived from microbes are crucial innovations that would help in reaching sustainability development goals worldwide while achieving bioeconomic growth. Trichoderma species are well-studied model fungal organisms used for their biocontrol properties with great potential to alleviate the use of agrochemicals in agriculture. However, identifying and characterizing effective natural products in novel species or strains as biological control products remains a meticulous process with many known challenges to be navigated. Integration of recent advancements in various "omics" technologies, next generation biodesign, machine learning, and artificial intelligence approaches could greatly advance bioprospecting goals. Herein, we propose a roadmap for assessing the potential impact of already known or newly discovered Trichoderma species for biocontrol applications. By screening publicly available Trichoderma genome sequences, we first highlight the prevalence of putative biosynthetic gene clusters and antimicrobial peptides among genomes as an initial step toward predicting which organisms could increase the diversity of natural products. Next, we discuss high-throughput methods for screening organisms to discover and characterize natural products and how these findings impact both fundamental and applied research fields.

Whole-Genome Sequencing and Bioinformatic Analysis of Environmental, Agricultural, and Human Campylobacter jejuni Isolates From East Tennessee.

As a leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter jejuni has a significant impact on human health in both the developed and developing worlds. Despite its prevalence as a human pathogen, the source of these infections remains poorly understood due to the mutation frequency of the organism and past limitations of whole genome analysis. Recent advances in both whole genome sequencing and computational methods have allowed for the high-resolution analysis of intraspecies diversity, leading multiple groups to postulate that these approaches may be used to identify the sources of Campylobacter jejuni infection. To address this hypothesis, our group conducted a regionally and temporally restricted sampling of agricultural and environmental Campylobacter sources and compared isolated C. jejuni genomes to those that caused human infections in the same region during the same time period. Through a network analysis comparing genomes from various sources, we found that human C. jejuni isolates clustered with those isolated from cattle and chickens, indicating these as potential sources of human infection in the region.

The RNase P family.

Ribonuclease P (RNase P) is a ribonucleoprotein comprised of a catalytic RNA subunit and one or several protein subunits. RNase P is best known for its role in 5'-processing of tRNA precursors. RNase P enzymes from almost all forms of life, including protein-synthesizing organelles, contain an RNase P with a conserved, homologous RNA. Five distinct structure classes of RNase P RNAs have been identified in bacteria and archaea; eukaryotic RNase P RNAs are not yet sufficiently well surveyed for structure classes to be defined. Here we will examine the structure variations in RNase P RNAs in bacteria, archaea, eukaryotes, plastids and mitochondria with special emphasis on the functional roles these unique secondary structures perform.

Genes within genes within bacteria.

Recently, an unusual gene structure has been described in species of the genus Thermus, in which the rpmH (ribosomal protein L34) coding sequence was found to be entirely overlapped by the unusually large rnpA (RNase P protein subunit) sequence. Gene overlap is common in viruses, but has not been seen to this extent in any bacterium.

Isolation and Whole-Genome Sequencing of Environmental Campylobacter.

As a leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter has a significant impact on human health. In the developed world, most campylobacteriosis cases are attributed to the consumption of undercooked, contaminated poultry; however, it has been shown that Campylobacter can be transmitted to humans through contaminated water and other types of food, including beef and milk. As such, high-resolution microbial source-tracking is essential for health department officials to determine the source(s) of Campylobacter outbreaks. For these reasons, this protocol provides the techniques needed for isolation of Campylobacter from agricultural and environmental sources, as well as human clinical specimens. Additionally, we describe a simple method for preparing high-quality genomic DNA that can be used for whole-genome sequencing and downstream bioinformatics analyses of Campylobacter genotypes. © 2018 by John Wiley & Sons, Inc.