Initially using 143 genomes, we developed a method for calculating the pair-wise distance between prokaryotic genomes using a Monte Carlo method to estimate the conservation of gene order. The method was based on repeatedly selecting five or six non-adjacent random orthologs from each of two genomes and determining if the chosen orthologs were in the same order. The raw distances were then corrected for gene order convergence using an adaptation of the Jukes-Cantor model, as well as using the common distance correction D' = -ln(1-D). First, we compared the distances found via the order of six orthologs to distances found based on ortholog gene content and small subunit rRNA sequences. The Jukes-Cantor gene order distances are reasonably well correlated with the divergence of rRNA (R (2) = 0.24), especially at rRNA Jukes-Cantor distances of less than 0.2 (R (2) = 0.52). Gene content is only weakly correlated with rRNA divergence (R (2) = 0.04) over all distances, however, it is especially strongly correlated at rRNA Jukes-Cantor distances of less than 0.1 (R (2) = 0.67). This initial work suggests that gene order may be useful in conjunction with other methods to help understand the relatedness of genomes. Using the gene order distances in 143 genomes, the relations of prokaryotes were studied using neighbor joining and agreement subtrees. We then repeated our study of the relations of prokaryotes using gene order in 172 complete genomes better representing a wider-diversity of prokaryotes. Consistently, our trees show the Actinobacteria as a sister group to the bulk of the Firmicutes. In fact, the robustness of gene order support was found to be considerably greater for uniting these two phyla than for uniting any of the proteobacterial classes together. The results are supportive of the idea that Actinobacteria and Firmicutes are closely related, which in turn implies a single origin for the gram-positive cell.

The selectivity of metal sensors for a single metal ion is critical for cellular metal homeostasis. A suite of metal-responsive regulators is required to maintain a prescribed balance of metal ions ensuring that each apo-protein binds the correct metal. However, there are cases when non-essential metals ions disrupt proper metal sensing. An analysis of the Ni-responsive transcriptome of the green alga Chlamydomonas reinhardtii reveals that Ni artificially turns on the CRR1-dependent Cu-response regulon. Since this regulon also responds to hypoxia, a combinatorial transcriptome analysis was leveraged to gain insight into the mechanisms by which Ni interferes with the homeostatic regulation of Cu and oxygen status. Based on parallels with the effect of Ni on the hypoxic response in animals, we propose that a possible link between Cu, oxygen and Ni sensing is an as yet uncharacterized prolyl hydroxylase that regulates a co-activator of CRR1. This analysis also identified transcriptional responses to the pharmacological activation of the Cu-deficiency regulon. Although the Ni-responsive CRR1 regulon is composed of 56 genes (defined as the primary response), 259 transcripts responded to Ni treatment only when a copy of the wild-type CRR1 gene was present. The genome-wide impact of CRR1 target genes on the transcriptome was also evident from the 210 transcripts that were at least 2-fold higher in the crr1 strain, where the abundance of many CRR1 targets was suppressed. Additionally, we identified 120 transcripts that responded to Ni independent of CRR1 function. The putative functions of the proteins encoded by these transcripts suggest that high Ni results in protein damage.

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.

Chlamydomonas reinhardtii is a unicellular chlorophyte alga that is widely studied as a reference organism for understanding photosynthesis, sensory and motile cilia, and for development of an algal-based platform for producing biofuels and bio-products. Its highly repetitive, ~205-kbp circular chloroplast genome and ~15.8-kbp linear mitochondrial genome were sequenced prior to the advent of high-throughput sequencing technologies. Here, high coverage shotgun sequencing was used to assemble both organellar genomes de novo. These new genomes correct dozens of errors in the prior genome sequences and annotations. Genome sequencing coverage indicates that each cell contains on average 83 copies of the chloroplast genome and 130 copies of the mitochondrial genome. Using protocols and analyses optimized for organellar transcripts, RNA-Seq was used to quantify their relative abundances across 12 different growth conditions. Forty-six percent of total cellular mRNA is attributable to high expression from a few dozen chloroplast genes. RNA-Seq data were used to guide gene annotation, to demonstrate polycistronic gene expression, and to quantify splicing of psaA and psbA introns. In contrast to a conclusion from a recent study, we found that chloroplast transcripts are not edited. Unexpectedly, cytosine-rich polynucleotide tails were observed at the 3'-end of all mitochondrial transcripts. A comparative genomics analysis of eight laboratory strains and 11 wild isolates of C. reinhardtii identified 2658 variants in the organellar genomes, which is 1/10th as much genetic diversity as is found in the nucleus.

In the original version of this Article, the y-axis label of Fig. 1d was incorrectly given as “Relative H3Ac/H3 levels” and should read “Relative H4Ac/H3 levels”. In addition, the probe target in the left panel of Figure 1f was misspelled as “Guliver” and should read “Gulliver”. Furthermore, the last label of the diagram on the top of Fig. 2c was given incorrectly as “PF26” and should read “PF25”. Moreover, the label of the branch pointed by the arrow symbol in cluster II of the phylogenetic tree in Fig. 3c was incorrectly labelled as “CrSRTb” and the correct label is “CrSRTB”. These errors have been corrected in both the PDF and HTML versions of the Article.

Oak represents a valuable natural resource across Northern Hemisphere ecosystems, attracting a large research community studying its genetics, ecology, conservation, and management. Here we introduce a draft genome assembly of valley oak (Quercus lobata) using Illumina sequencing of adult leaf tissue of a tree found in an accessible, well-studied, natural southern California population. Our assembly includes a nuclear genome and a complete chloroplast genome, along with annotation of encoded genes. The assembly contains 94,394 scaffolds, totaling 1.17 Gb with 18,512 scaffolds of length 2 kb or longer, with a total length of 1.15 Gb, and a N50 scaffold size of 278,077 kb. The k-mer histograms indicate an diploid genome size of ∼720-730 Mb, which is smaller than the total length due to high heterozygosity, estimated at 1.25%. A comparison with a recently published European oak (Q. robur) nuclear sequence indicates 93% similarity. The Q. lobata chloroplast genome has 99% identity with another North American oak, Q. rubra Preliminary annotation yielded an estimate of 61,773 predicted protein-coding genes, of which 71% had similarity to known protein domains. We searched 956 Benchmarking Universal Single-Copy Orthologs, and found 863 complete orthologs, of which 450 were present in > 1 copy. We also examined an earlier version (v0.5) where duplicate haplotypes were removed to discover variants. These additional sources indicate that the predicted gene count in Version 1.0 is overestimated by 37-52%. Nonetheless, this first draft valley oak genome assembly represents a high-quality, well-annotated genome that provides a tool for forest restoration and management practices.

Silencing of exogenous DNA can make transgene expression very inefficient. Genetic screens in the model alga Chlamydomonas have demonstrated that transgene silencing can be overcome by mutations in unknown gene(s), thus producing algal strains that stably express foreign genes to high levels. Here, we show that the silencing mechanism specifically acts on transgenic DNA. Once a permissive chromatin structure has assembled, transgene expression can persist even in the absence of mutations disrupting the silencing pathway. We have identified the gene conferring the silencing and show it to encode a sirtuin-type histone deacetylase. Loss of gene function does not appreciably affect endogenous gene expression. Our data suggest that transgenic DNA is recognized and then quickly inactivated by the assembly of a repressive chromatin structure composed of deacetylated histones. We propose that this mechanism may have evolved to provide protection from potentially harmful types of environmental DNA.

Background

A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity.

Results

We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages.

Conclusions

Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

The genus Quercus, which emerged ∼55 million years ago during globally warm temperatures, diversified into ∼450 extant species. We present a high-quality de novo genome assembly of a California endemic oak, Quercus lobata, revealing features consistent with oak evolutionary success. Effective population size remained large throughout history despite declining since early Miocene. Analysis of 39,373 mapped protein-coding genes outlined copious duplications consistent with genetic and phenotypic diversity, both by retention of genes created during the ancient γ whole genome hexaploid duplication event and by tandem duplication within families, including numerous resistance genes and a very large block of duplicated DUF247 genes, which have been found to be associated with self-incompatibility in grasses. An additional surprising finding is that subcontext-specific patterns of DNA methylation associated with transposable elements reveal broadly-distributed heterochromatin in intergenic regions, similar to grasses. Collectively, these features promote genetic and phenotypic variation that would facilitate adaptability to changing environments.

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.