Ribosomal RNA Operon Research Articles

Trade-off between microbial ecophysiological features regulated by soil fertility governs plant residue decomposition

Soil fertility influences microbial-driven plant residue decomposition, thus further affecting soil organic carbon (C) formation. Both ecological and physiological features of microbial communities change with soil fertilities, however, the mechanism of how soil fertility influences microbial ecophysiological features (both the taxonomic and functional profiles) driving plant residue decomposition is unclear. Here, four series of microcosms were set up, inoculated with two distinct soils, each with low and high fertility levels as a result of contrasting long-term fertilization regimes, and incubated with 13C-labeled rice straw amendments. DNA stable-isotope probing (DNA-SIP) based amplicon sequencing and shotgun metagenomic sequencing were conducted. We found that both bacterial community composition (including the relative abundance, α- and β-diversities, and taxonomic composition) and carbohydrate metabolic efficiency were significantly changed under different soil fertilities. High fertile soils gave rise to greater increases in bacterial populations and straw decomposition than low fertile soils, yet DNA-SIP-based shotgun metagenomic sequencing showed that the bacterial average carbohydrate metabolic efficiency, characterized by the average carbohydrate-active enzyme (CAZyme) abundance within each bacterial phylum was significantly lower. Linear regressions between bacterial ribosomal RNA operon (rrn) copy number and bacterial carbohydrate metabolic efficiency under contrasting soil fertilities suggested that high fertile soils select for fast-but-inefficient bacteria to degrade rice straw while low fertile soils select for slow-but-efficient ones. Collectively, the data demonstrate a fundamental trade-off between the ecological and physiological attributes of straw-degrading microbial communities. The trade-off is regulated by soil fertility and in turn governs plant residue decomposition. This work provides novel insights for better understanding the ecological and physiological roles of microbes in organic matter decomposition and global terrestrial C cycling, especially in the context of crop straw degradation under contrasting soil fertilities.

Nisin E, a New Nisin Variant Produced by Streptococcus equinus MDC1

Members of the genus Streptococcus inhabit a variety of sites in humans and other animals and some species are prolific producers of proteinaceous antibiotics (bacteriocins). As little is known about (i) streptococci inhabiting domestic pets, and (ii) whether novel bacteriocin-producing streptococci can be isolated from domestic pets, the aim of this study is to address these gaps in the research literature. In this study, Streptococcus equinus MDC1, isolated from a healthy dog, was found to exhibit potent antibacterial activity against Micrococcus luteus in a simultaneous antagonism assay, suggesting that strain MDC1 produces a lantibiotic bacteriocin. The inhibitory activity spectrum of S. equinus MDC1, determined using agar-based deferred antagonism assays against >70 indicator strains, was found to be similar to that of nisin U (a lantibiotic produced by Streptococcus uberis). However, the spectra of the two bacteriocins differed by 23 strains, mainly with the MDC1 bacteriocin having no inhibitory activity towards certain streptococci of human origin (e.g., Streptococcus gordonii, Streptococcus anginosus, Streptococcus salivarius). The genome of S. equinus MDC1, which was sequenced completely using single-molecule real-time (SMRT) next-generation DNA sequencing technology, comprises a single 1,936,555-basepair chromosome containing seven copies of the ribosomal RNA operon, 69 tRNA genes and nearly 1900 putative coding sequences. Analysis of the MDC1 genome sequence using the bacteriocin detection algorithms BAGEL4 and antiSMASH revealed the location of a 13,164-basepair 11-gene locus, designated nmd, which encoded a mature nisin E peptide that differed from nisin U by only two amino acids (Ile15→Ala and Leu21→Ile) and an extra C-terminal asparagine residue, and the proteins required for post-translational modification of the bacteriocin, processing, export, and producer immunity. Despite the high homology (90.6% identity, 93.8% similarity) between nisin E and nisin U, there was considerably less homology (47.4–76.3% identity, 68.4–88.8% similarity) between the other proteins encoded by their respective biosynthetic loci. This new natural variant of nisin, called nisin E, represents the first nisin variant to be reported for S. equinus; additionally, its differences with nisin U may provide some insight into the amino acids that influence bacteriocin potency and killing spectrum.

A high-quality genome compendium of the human gut microbiome of Inner Mongolians.

Metagenome-based resources have revealed the diversity and function of the human gut microbiome, but further understanding is limited by insufficient genome quality and a lack of samples from typically understudied populations. Here we used hybrid long-read PromethION and short-read HiSeq sequencing to characterize the faecal microbiota of 60 Inner Mongolian individuals (n = 180 samples over three time points) who were part of a probiotic yogurt intervention trial. We present the Inner Mongolian Gut Genome catalogue, comprising 802 closed and 5,927 high-quality metagenome-assembled genomes. This approach achieved high genome continuity and substantially increased the resolution of genomic elements, including ribosomal RNA operons, metabolic gene clusters, prophages and insertion sequences. Particularly, we report the ribosomal RNA operon copy numbers for uncultured species, over 12,000 previously undescribed gut prophages and the distribution of insertion sequence elements across gut bacteria. Overall, these data provide a high-quality, large-scale resource for studying the human gut microbiota.

Evaluation of bacterial consortia associated with dairy fermentation by ribosomal RNA (rrn) operon metabarcoding strategy using MinION device

The ability of the third generation sequencing technologies to provide longer sequence reads contributes to the use of the longest possible amplicons as specific bacterial markers for metabarcoding studies. Nanopore sequencing technologies are increasingly used worldwide to profile microbiomes in environmental and food samples. The identification of beneficial or pathogenic bacteria in dairy fermented foods is related to their valuable health properties and also contributes to food safety issues. Here we described and optimised a PCR-based methodology of almost the entire ribosomal operon sequences (16S-ITS-23S) and their subsequent sequencing by MinION device. We used three different sequencing data processing and analysis strategies. Two of those utilized user-friendly software without the need of being conversant with any programming language. We tested all workflows on a simple mock community composed of a mixture of 7 bacterial DNA. Our scripted bioinformatics pipeline denoted as “AEROS”, representing an approach based on taxonomic classification with our reference database called AEROS-DB (Almost Entire Ribosomal Operon Sequences), was applied to traditional Slovak sheep cheese made from unpasteurized milk. All bacterial genera included in the mock community were detected with relatively small differences compared to the expected relative abundance using each of the three approaches. The AEROS approach provided more accurate composition data on this community at the species level as well. The results suggested that the use of almost entire rrn operon sequences in metabarcoding studies is suitable to analyze the bacterial consortia in cheeses and related dairy fermented products.

Improved microbial genomes and gene catalog of the chicken gut from metagenomic sequencing of high-fidelity long reads.

Due to the importance of chicken production and the remarkable influence of the gut microbiota on host health and growth, tens of thousands of metagenome-assembled genomes (MAGs) have been constructed for the chicken gut microbiome. However, due to the limitations of short-read sequencing and assembly technologies, most of these MAGs are far from complete, are of lower quality, and include contaminant reads. We generated 332 Gb of high-fidelity (HiFi) long reads from the 5 chicken intestinal compartments and assembled 461 and 337 microbial genomes, of which 53% and 55% are circular, at the species and strain levels, respectively. For the assembled microbial genomes, approximately 95% were regarded as complete according to the "RNA complete" criteria, which requires at least 1 full-length ribosomal RNA (rRNA) operon encoding all 3 types of rRNA (16S, 23S, and 5S) and at least 18 copies of full-length transfer RNA genes. In comparison with the short-read-derived chicken MAGs, 384 (83% of 461) and 89 (26% of 337) strain-level and species-level genomes in this study are novel, with no matches to previously reported sequences. At the gene level, one-third of the 2.5 million genes in the HiFi-derived gene catalog are novel and cannot be matched to the short-read-derived gene catalog. Moreover, the HiFi-derived genomes have much higher continuity and completeness, as well as lower contamination; the HiFi-derived gene catalog has a much higher ratio of complete gene structures. The dominant phylum in our HiFi-assembled genomes was Firmicutes (82.5%), and the foregut was highly enriched in 5 genera: Ligilactobacillus, Limosilactobacillus, Lactobacillus, Weissella, and Enterococcus, all of which belong to the order Lactobacillales. Using GTDB-Tk, all 337 species-level genomes were successfully classified at the order level; however, 2, 35, and 189 genomes could not be classified into any known family, genus, and species, respectively. Among these incompletely classified genomes, 9 and 49 may belong to novel genera and species, respectively, because their 16S rRNA genes have identities lower than 95% and 97% to any known 16S rRNA genes. HiFi sequencing not only produced metagenome assemblies and gene structures with markedly improved quality but also recovered a substantial portion of novel genomes and genes that were missed in previous short-read-based metagenome studies. The novel genomes and species obtained in this study will facilitate gut microbiome and host-microbiota interaction studies, thereby contributing to the sustainable development of poultry resources.

Ribosomal RNA operons define a central functional compartment in the Streptomyces chromosome.

Streptomyces are prolific producers of specialized metabolites with applications in medicine and agriculture. These bacteria possess a large linear chromosome genetically compartmentalized: core genes are grouped in the central part, while terminal regions are populated by poorly conserved genes. In exponentially growing cells, chromosome conformation capture unveiled sharp boundaries formed by ribosomal RNA (rrn) operons that segment the chromosome into multiple domains. Here we further explore the link between the genetic distribution of rrn operons and Streptomyces genetic compartmentalization. A large panel of genomes of species representative of the genus diversity revealed that rrn operons and core genes form a central skeleton, the former being identifiable from their core gene environment. We implemented a new nomenclature for Streptomyces genomes and trace their rrn-based evolutionary history. Remarkably, rrn operons are close to pericentric inversions. Moreover, the central compartment delimited by rrn operons has a very dense, nearly invariant core gene content. Finally, this compartment harbors genes with the highest expression levels, regardless of gene persistence and distance to the origin of replication. Our results highlight that rrn operons are structural boundaries of a central functional compartment prone to transcription in Streptomyces.

Spectinomycin resistance in Lysobacter enzymogenes is due to its rRNA target but also relies on cell-wall recycling and purine biosynthesis

Resistance to spectinomycin emerged after widely used for treatment of gonorrhea. Previous studies revealed that Lysobacter enzymogenes strain C3 (LeC3) exhibited elevated level of intrinsic resistance to spectinomycin. In this study, we screened a Tn5 transposon mutant library of LeC3 to elucidate the underlying molecular mechanisms of spectinomycin resistance. Insertion sites in 15 out of 19 mutants recovered with decreased spectinomycin resistance were located on two ribosomal RNA operons at different loci, indicating the pivotal role of ribosomal RNAs in conferring spectinomycin resistance in L. enzymogenes. The other mutants harbored mutations in the tuf, rpoD, mltB, and purB genes. Among them, the tuf and rpoD genes, respectively, encode a translation elongation factor Tu and an RNA polymerase primary sigma factor. They both contribute to protein biosynthesis, where ribosomal RNAs play essential roles. The mltB gene, whose product is involved in cell-wall recycling, was not only associated with resistance against spectinomycin, but also conferred resistance to osmotic stress and ampicillin. In addition, mutation of the purB gene, for which its product is involved in the biosynthesis of inosine and adenosine monophosphates, led to decreased spectinomycin resistance. Addition of exogenous adenine at lower concentration in medium restored the growth deficiency in the purB mutant and increased bacterial resistance to spectinomycin. These results suggest that while cell-wall recycling and purine biosynthesis might contribute to spectinomycin resistance, target rRNAs play critical role in spectinomycin resistance in L. enzymogenes.

Characterization of the pathogenicity of a Bacillus cereus isolate from the Mariana Trench

ABSTRACT Bacillus cereus is an important opportunistic pathogen widely distributed in the environment. In this study, we reported the isolation and characterization of a B. cereus isolate, MB1, from the Challenger Deep of the Mariana Trench. MB1 is aerobic, motile, and able to form endospores. It possesses 5966 genes distributed on a circular chromosome and two plasmids. The MB1 genome contains 14 sets of 23S, 5S, and 16S ribosomal RNA operons, 106 tRNA genes, 4 sRNA genes, 12 genomic islands, 13 prophages, and 302 putative virulence genes, including enterotoxins and cytolysins. Infection studies showed that MB1 was able to cause acute and lethal infection in fish and mice, and was highly toxic to mammalian cells. MB1 induced, in a dose-dependent manner, pyroptotic cell death, characterized by activation of caspase-1, cleavage of gasdermin D, and release of IL-1β and IL-18. MB1 spores exhibited swimming and haemolytic capacity, but were severely attenuated in pathogenicity, which, however, was regained to the full extent when the spores germinated under suitable conditions. Taken together, these results provide new insights into the biological and pathogenic mechanism of deep sea B. cereus.

Substrate Utilization and Competitive Interactions Among Soil Bacteria Vary With Life-History Strategies.

Microorganisms have evolved various life-history strategies to survive fluctuating resource conditions in soils. However, it remains elusive how the life-history strategies of microorganisms influence their processing of organic carbon, which may affect microbial interactions and carbon cycling in soils. Here, we characterized the genomic traits, exometabolite profiles, and interactions of soil bacteria representing copiotrophic and oligotrophic strategists. Isolates were selected based on differences in ribosomal RNA operon (rrn) copy number, as a proxy for life-history strategies, with pairs of “high” and “low” rrn copy number isolates represented within the Micrococcales, Corynebacteriales, and Bacillales. We found that high rrn isolates consumed a greater diversity and amount of substrates than low rrn isolates in a defined growth medium containing common soil metabolites. We estimated overlap in substrate utilization profiles to predict the potential for resource competition and found that high rrn isolates tended to have a greater potential for competitive interactions. The predicted interactions positively correlated with the measured interactions that were dominated by negative interactions as determined through sequential growth experiments. This suggests that resource competition was a major force governing interactions among isolates, while cross-feeding of metabolic secretion likely contributed to the relatively rare positive interactions observed. By connecting bacterial life-history strategies, genomic features, and metabolism, our study advances the understanding of the links between bacterial community composition and the transformation of carbon in soils.

RRNA operon multiplicity as a bacterial genome stability insurance policy.

Quick growth restart after upon encountering favourable environmental conditions is a major fitness contributor in natural environment. It is widely assumed that the time required to restart growth after nutritional upshift is determined by how long it takes for cells to synthesize enough ribosomes to produce the proteins required to reinitiate growth. Here we show that a reduction in the capacity to synthesize ribosomes by reducing number of ribosomal RNA (rRNA) operons (rrn) causes a longer transition from stationary phase to growth of Escherichia coli primarily due to high mortality rates. Cell death results from DNA replication blockage and massive DNA breakage at the sites of the remaining rrn operons that become overloaded with RNA polymerases (RNAPs). Mortality rates and growth restart duration can be reduced by preventing R-loop formation and improving DNA repair capacity. The same molecular mechanisms determine the duration of the recovery phase after ribosome-damaging stresses, such as antibiotics, exposure to bile salts or high temperature. Our study therefore suggests that a major function of rrn operon multiplicity is to ensure that individual rrn operons are not saturated by RNAPs, which can result in catastrophic chromosome replication failure and cell death during adaptation to environmental fluctuations.

Prokaryotic diversity in marine and freshwater recirculating aquaculture systems

AbstractIntensive aquaculture food production has been continually increasing over the past decades. Recirculating aquaculture is an innovative technology which relies on water reuse and removal of toxic metabolites by utilizing specific prokaryotic groups such as nitrifiers, settled in special biofilters. Focused on recent next generation sequencing‐based studies on the microbiome of recirculating aquaculture systems, this review highlights dominant genera and families, and investigates their possible metabolic function in key niches within recirculating aquaculture systems (rearing water, biofilters and fish mucosal microbiome), while aiming to identify core prokaryotic groups across studies. A total of 136 genera belonging to 77 families and 13 phyla have been characterized as dominant or functionally important in relevant studies, mainly isolated from nitrification biofilters, system water and intestinal samples of the farmed species. Proteobacteria is undoubtedly the most abundant phylum, followed by Bacteroidetes, Firmicutes and Actinobacteria, while Rhodobacteriaceae and Flavobacteriaceae are the common dominant families. Nitrosomonadaceae and Nitrospira members are enriched in biofilter samples and serve as the most common ammonium and nitrite oxidizers, respectively. Composition of dominant genera appears system specific due to unique characteristics of individual RAS designs, although faint community patterns emerge when examining RAS by their salinity level. Considering the number of ribosomal RNA operon copies, 75% of genera are likely slow growers, dominating biofilter and water communities, whereas the few fast‐growing opportunists are mainly associated with the intestine of farmed species. Finally, this review stresses the lack of information regarding archaeal and microeukaryotic communities in recirculating aquaculture systems and prokaryotic–protist interactions.

Long-read sequencing for identification of insertion sites in large transposon mutant libraries

Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long-Read Transposon Insertion-site Sequencing). LoRTIS enabled the unique localisation of transposon insertion sites within long repetitive genetic elements of E. coli, such as the transposase genes of insertion sequences and copies of the ~ 5 kb ribosomal RNA operon. We demonstrate that LoRTIS is reproducible, gives comparable results to short-read TIS methods for essential genes, and better resolution around repeat elements. The Oxford Nanopore sequencing device that we used is cost-effective, small and easily portable. Thus, LoRTIS is an efficient means of uniquely identifying transposon insertion sites within long repetitive genetic elements and can be easily transported to, and used in, laboratories that lack access to expensive DNA sequencing facilities.

Regulatory perturbations of ribosome allocation in bacteria reshape the growth proteome with a trade-off in adaptation capacity

SummaryBacteria regulate their cellular resource allocation to enable fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth, and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655. Mutants with only three of the seven copies of ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied fast-growing rpoB mutants. Comparing these two different regulatory perturbations (rRNA promoters or rpoB mutations), we show how they reshape the proteome for growth with a concomitant fitness cost. The fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. They showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments.

Identification of Pentatrichomonas hominis in preputial washes of bulls in Brazil.

The parabasalid Pentatrichomonas hominis is generally considered to represent a symbiotic component of the gastrointestinal microbiota in a wide variety of vertebrate hosts including humans. Nevertheless, a limited number of studies have raised the possibility that it may act as a pathogen of humans, dogs, and pigs and that some human infections may have a zoonotic origin. Data from North America revealed an association between P. hominis and the bovine urogenital tract, principally in bulls and rarely in cows. The importance of this observation is linked to possible interference in the accurate diagnosis of the economically important venereal pathogen Tritrichomonas foetus. The current study employed culture-based and molecular methods to examine the preputial cavities of four breeding bulls, raised in open pasture in southeastern Brazil, for the presence of trichomonads. Motile protozoa were isolated from three of the bulls and were definitively identified as P. hominis based on nucleotide sequencing of polymerase chain reaction (PCR) amplicons derived from the ribosomal RNA operon (ITS1-5.8S rDNA-ITS2) of the parasite. The potential implications of these findings for bovine and human health are discussed.

Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli.

The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae, a Bacteroidia which rarely does. In E. coli, 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli. In F. johnsoniae, deletion of three ribosomal RNA (rrn) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli, consistent with SD usage in the two organisms.

Long-read sequencing to interrogate strain-level variation among adherent-invasive Escherichia coli isolated from human intestinal tissue.

Adherent-invasive Escherichia coli (AIEC) is a pathovar linked to inflammatory bowel diseases (IBD), especially Crohn’s disease, and colorectal cancer. AIEC are genetically diverse, and in the absence of a universal molecular signature, are defined by in vitro functional attributes. The relative ability of difference AIEC strains to colonize, persist, and induce inflammation in an IBD-susceptible host is unresolved. To evaluate strain-level variation among tissue-associated E. coli in the intestines, we develop a long-read sequencing approach to identify AIEC by strain that excludes host DNA. We use this approach to distinguish genetically similar strains and assess their fitness in colonizing the intestine. Here we have assembled complete genomes using long-read nanopore sequencing for a model AIEC strain, NC101, and seven strains isolated from the intestinal mucosa of Crohn’s disease and non-Crohn’s tissues. We show these strains can colonize the intestine of IBD susceptible mice and induce inflammatory cytokines from cultured macrophages. We demonstrate that these strains can be quantified and distinguished in the presence of 99.5% mammalian DNA and from within a fecal population. Analysis of global genomic structure and specific sequence variation within the ribosomal RNA operon provides a framework for efficiently tracking strain-level variation of closely-related E. coli and likely other commensal/pathogenic bacteria impacting intestinal inflammation in experimental settings and IBD patients.

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.

A method for achieving complete microbial genomes and improving bins from metagenomics data.

Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete genomes from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Some studies have attempted to extract complete bacterial, archaeal, and viral genomes and often focus on species with circular genomes so they can help confirm completeness with circularity. However, less than 100 circularized bacterial and archaeal genomes have been assembled and published from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a semi-automated method called Jorg to help circularize small bacterial, archaeal, and viral genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one ribosomal RNA operon. In addition to 34 circular CPR genomes, we present one circular Margulisbacteria genome, one circular Chloroflexi genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA. Code and a tutorial for this method is available at https://github.com/lmlui/Jorg and is available on the DOE Systems Biology KnowledgeBase as a beta app.

Complete Genome Sequence of Cellulomonas sp. JZ18, a Root Endophytic Bacterium Isolated from the Perennial Desert Tussock-Grass Panicum turgidum

Cellulomonas sp. JZ18 is a gram-positive, rod shaped bacterium that was previously isolated from the root endosphere of the perennial desert tussock-grass Panicum turgidum. Genome coverage of PacBio sequencing was approximately 199X. Genome assembly generated a single chromosome of 7,421,843 base pairs with a guanine-cytosine (GC) content of 75.60% with 3240 protein coding sequences, 361 pseudo genes, three ribosomal RNA operons, three non-coding RNAs and 45 transfer RNAs. Comparison of JZ18's genome with type strains from the same genus, using digital DNA-DNA hybridization and average nucleotide identity calculations, revealed that JZ18 might potentially belong to a new species. Functional analysis revealed the presence of genes that may complement previously observed biochemical and plant phenotypes. Furthermore, the presence of a number of enzymes could be of potential use in industrial processes as biocatalysts. Genome sequencing and analysis, coupled with comparative genomics, of endophytic bacteria for their potential plant growth promoting activities under different soil conditions will accelerate the knowledge and applications of biostimulants in sustainable agriculture.

The Antibiotic Dosage of Fastest Resistance Evolution: Gene Amplifications Underpinning the Inverted-U.

To determine the dosage at which antibiotic resistance evolution is most rapid, we treated Escherichia coli in vitro, deploying the antibiotic erythromycin at dosages ranging from zero to high. Adaptation was fastest just below erythromycin’s minimal inhibitory concentration (MIC) and genotype-phenotype correlations determined from whole genome sequencing revealed the molecular basis: simultaneous selection for copy number variation in three resistance mechanisms which exhibited an “inverted-U” pattern of dose-dependence, as did several insertion sequences and an integron. Many genes did not conform to this pattern, however, reflecting changes in selection as dose increased: putative media adaptation polymorphisms at zero antibiotic dosage gave way to drug target (ribosomal RNA operon) amplification at mid dosages whereas prophage-mediated drug efflux amplifications dominated at the highest dosages. All treatments exhibited E. coli increases in the copy number of efflux operons acrAB and emrE at rates that correlated with increases in population density. For strains where the inverted-U was no longer observed following the genetic manipulation of acrAB, it could be recovered by prolonging the antibiotic treatment at subMIC dosages.