"Candidatus Intestinibacterium parameciiphilum"-member of the "Candidatus Paracaedibacteraceae" family (Alphaproteobacteria, Holosporales) inhabiting the ciliated protist Paramecium.

Protists frequently host diverse bacterial symbionts, in particular those affiliated with the order Holosporales (Alphaproteobacteria). All characterised members of this bacterial lineage have been retrieved in obligate association with a wide range of eukaryotes, especially multiple protist lineages (e.g. amoebozoans, ciliates, cercozoans, euglenids, and nucleariids), as well as some metazoans (especially arthropods and related ecdysozoans). While the genus Paramecium and other ciliates have been deeply investigated for the presence of symbionts, known members of the family "Candidatus Paracaedibacteraceae" (Holosporales) are currently underrepresented in such hosts. Herein, we report the description of "Candidatus Intestinibacterium parameciiphilum" within the family "Candidatus Paracaedibacteraceae", inhabiting the cytoplasm of Paramecium biaurelia. This novel bacterium is almost twice as big as its relative "Candidatus Intestinibacterium nucleariae" from the opisthokont Nuclearia and does not present a surrounding halo. Based on phylogenetic analyses of 16S rRNA gene sequences, we identified six further potential species-level lineages within the genus. Based on the provenance of the respective samples, we investigated the environmental distribution of the representatives of "Candidatus Intestinibacterium" species. Obtained results are consistent with an obligate endosymbiotic lifestyle, with protists, in particular freshwater ones, as hosts. Thus, available data suggest that association with freshwater protists could be the ancestral condition for the members of the "Candidatus Intestinibacterium" genus.

Dynamics of Gene Loss following Ancient Whole-Genome Duplication in the Cryptic Paramecium Complex.

Whole-genome duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages. The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods, and the relative contributions of different selective pressures to their maintenance are still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliate Paramecium tetraurelia and two of its sister species from the Paramecium aurelia complex. Here, we report the genome sequence and analysis of 10 additional P. aurelia species and 1 additional out group, revealing aspects of post-WGD evolution in 13 species sharing a common ancestral WGD. Contrary to the morphological radiation of vertebrates that putatively followed two WGD events, members of the cryptic P. aurelia complex have remained morphologically indistinguishable after hundreds of millions of years. Biases in gene retention compatible with dosage constraints appear to play a major role opposing post-WGD gene loss across all 13 species. In addition, post-WGD gene loss has been slower in Paramecium than in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong in Paramecium. A near complete lack of recent single-gene duplications in Paramecium provides additional evidence for strong selective pressures against gene dosage changes. This exceptional data set of 13 species sharing an ancestral WGD and 2 closely related out group species will be a useful resource for future studies on Paramecium as a major model organism in the evolutionary cell biology.

A Population-Genetic Lens into the Process of Gene Loss Following Whole-Genome Duplication.

Whole-genome duplications (WGDs) have occurred in many eukaryotic lineages. However, the underlying evolutionary forces and molecular mechanisms responsible for the long-term retention of gene duplicates created by WGDs are not well understood. We employ a population-genomic approach to understand the selective forces acting on paralogs and investigate ongoing duplicate-gene loss in multiple species of Paramecium that share an ancient WGD. We show that mutations that abolish protein function are more likely to be segregating in retained WGD paralogs than in single-copy genes, most likely because of ongoing nonfunctionalization post-WGD. This relaxation of purifying selection occurs in only one WGD paralog, accompanied by the gradual fixation of nonsynonymous mutations and reduction in levels of expression, and occurs over a long period of evolutionary time, "marking" one locus for future loss. Concordantly, the fitness effects of new nonsynonymous mutations and frameshift-causing indels are significantly more deleterious in the highly expressed copy compared with their paralogs with lower expression. Our results provide a novel mechanistic model of gene duplicate loss following WGDs, wherein selection acts on the sum of functional activity of both duplicate genes, allowing the two to wander in expression and functional space, until one duplicate locus eventually degenerates enough in functional efficiency or expression that its contribution to total activity is too insignificant to be retained by purifying selection. Retention of duplicates by such mechanisms predicts long times to duplicate-gene loss, which should not be falsely attributed to retention due to gain/change in function.

Rates of Mutations and Transcript Errors in the Foodborne Pathogen Salmonella enterica subsp. enterica.

Because errors at the DNA level power pathogen evolution, a systematic understanding of the rate and molecular spectra of mutations could guide the avoidance and treatment of infectious diseases. We thus accumulated tens of thousands of spontaneous mutations in 768 repeatedly bottlenecked lineages of 18 strains from various geographical sites, temporal spread, and genetic backgrounds. Entailing over ∼1.36 million generations, the resultant data yield an average mutation rate of ∼0.0005 per genome per generation, with a significant within-species variation. This is one of the lowest bacterial mutation rates reported, giving direct support for a high genome stability in this pathogen resulting from high DNA-mismatch-repair efficiency and replication-machinery fidelity. Pathogenicity genes do not exhibit an accelerated mutation rate, and thus, elevated mutation rates may not be the major determinant for the diversification of toxin and secretion systems. Intriguingly, a low error rate at the transcript level is not observed, suggesting distinct fidelity of the replication and transcription machinery. This study urges more attention on the most basic evolutionary processes of even the best-known human pathogens and deepens the understanding of their genome evolution.

Chromosome organization and gene expansion in the highly fragmented genome of the ciliate Strombidium stylifer.

Chromosomes are well-organized carriers of genetic information in eukaryotes and are usually quite long, carrying hundreds and thousands of genes. Intriguingly, a clade of single-celled ciliates, Spirotrichea, feature nanochromosomes-also called "gene-sized chromosomes". These chromosomes predominantly carry only one gene, flanked by short telomere sequences. However, the organization and copy number variation of the chromosomes in these highly fragmented genomes remain unexplored in many groups of Spirotrichea, including the marine Strombidium. Using deep genome sequencing, we assembled the macronuclear genome of Strombidium stylifer into more than 18,000 nanochromosomes (~2.4 Kb long on average). Our results show that S. stylifer occupies an intermediate position during the evolutionary history of Strombidium lineage and experienced significant expansions in several gene families related to guanyl ribonucleotide binding. Based on the nucleotide distribution bias analysis and conserved motifs search in non-genic regions, we found that the subtelomeric regions have a conserved adenine-thymine (AT)-rich sequence motif. We also found that the copy number of nanochromosomes lacks precise regulation. This work sheds light on the unique features of chromosome structure in eukaryotes with highly fragmented genomes and reveals that a rather specialized evolutionary strategy at the genomic level has resulted in great diversity within the ciliated lineages.

Low Base-Substitution Mutation Rate but High Rate of Slippage Mutations in the Sequence Repeat-Rich Genome of Dictyostelium discoideum.

We describe the rate and spectrum of spontaneous mutations for the social amoeba Dictyostelium discoideum, a key model organism in molecular, cellular, evolutionary and developmental biology. Whole-genome sequencing of 37 mutation accumulation lines of D. discoideum after an average of 1,500 cell divisions yields a base-substitution mutation rate of 2.47 × 10-11 per site per generation, substantially lower than that of most eukaryotic and prokaryotic organisms, and of the same order of magnitude as in the ciliates Paramecium tetraurelia and Tetrahymena thermophila Known for its high genomic AT content and abundance of simple sequence repeats, we observe that base-substitution mutations in D. discoideum are highly A/T biased. This bias likely contributes both to the high genomic AT content and to the formation of simple sequence repeats in the AT-rich genome of Dictyostelium discoideum In contrast to the situation in other surveyed unicellular eukaryotes, indel rates far exceed the base-substitution mutation rate in this organism with a high proportion of 3n indels, particularly in regions without simple sequence repeats. Like ciliates, D. discoideum has a large effective population size, reducing the power of random genetic drift, magnifying the effect of selection on replication fidelity, in principle allowing D. discoideum to evolve an extremely low base-substitution mutation rate.

Estimation of the Genome-Wide Mutation Rate and Spectrum in the Archaeal Species Haloferax volcanii.

Organisms adapted to life in extreme habitats (extremophiles) can further our understanding of the mechanisms of genetic stability, particularly replication and repair. Despite the harsh environmental conditions they endure, these extremophiles represent a great deal of the Earth's biodiversity. Here, for the first time in a member of the archaeal domain, we report a genome-wide assay of spontaneous mutations in the halophilic species Haloferax volcanii using a direct and unbiased method: mutation accumulation experiments combined with deep whole-genome sequencing. H. volcanii is a key model organism not only for the study of halophilicity, but also for archaeal biology in general. Our methods measure the genome-wide rate, spectrum, and spatial distribution of spontaneous mutations. The estimated base substitution rate of 3.15 × 10-10 per site per generation, or 0.0012 per genome per generation, is similar to the value found in mesophilic prokaryotes (optimal growth at ∼20-45°). This study contributes to a comprehensive phylogenetic view of how evolutionary forces and molecular mechanisms shape the rate and molecular spectrum of mutations across the tree of life.

ADFinder: accurate detection of programmed DNA elimination using NGS high-throughput sequencing data.

MOTIVATION: Programmed DNA elimination (PDE) plays a crucial role in the transitions between germline and somatic genomes in diverse organisms ranging from unicellular ciliates to multicellular nematodes. However, software specific for the detection of DNA splicing events is scarce. In this paper, we describe Accurate Deletion Finder (ADFinder), an efficient detector of PDEs using high-throughput sequencing data. ADFinder can predict PDEs with relatively low sequencing coverage, detect multiple alternative splicing forms in the same genomic location and calculate the frequency for each splicing event. This software will facilitate research of PDEs and all down-stream analyses. RESULTS: By analyzing genome-wide DNA splicing events in two micronuclear genomes of Oxytricha trifallax and Tetrahymena thermophila, we prove that ADFinder is effective in predicting large scale PDEs. AVAILABILITY AND IMPLEMENTATION: The source codes and manual of ADFinder are available in our GitHub website: https://github.com/weibozheng/ADFinder. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Population Genetics of Paramecium Mitochondrial Genomes: Recombination, Mutation Spectrum, and Efficacy of Selection.

The evolution of mitochondrial genomes and their population-genetic environment among unicellular eukaryotes are understudied. Ciliate mitochondrial genomes exhibit a unique combination of characteristics, including a linear organization and the presence of multiple genes with no known function or detectable homologs in other eukaryotes. Here we study the variation of ciliate mitochondrial genomes both within and across 13 highly diverged Paramecium species, including multiple species from the P. aurelia species complex, with four outgroup species: P. caudatum, P. multimicronucleatum, and two strains that may represent novel related species. We observe extraordinary conservation of gene order and protein-coding content in Paramecium mitochondria across species. In contrast, significant differences are observed in tRNA content and copy number, which is highly conserved in species belonging to the P. aurelia complex but variable among and even within the other Paramecium species. There is an increase in GC content from ∼20% to ∼40% on the branch leading to the P. aurelia complex. Patterns of polymorphism in population-genomic data and mutation-accumulation experiments suggest that the increase in GC content is primarily due to changes in the mutation spectra in the P. aurelia species. Finally, we find no evidence of recombination in Paramecium mitochondria and find that the mitochondrial genome appears to experience either similar or stronger efficacy of purifying selection than the nucleus.

Escherichia coli cultures maintain stable subpopulation structure during long-term evolution.

How genetic variation is generated and maintained remains a central question in evolutionary biology. When presented with a complex environment, microbes can take advantage of genetic variation to exploit new niches. Here we present a massively parallel experiment where WT and repair-deficient (∆mutL) Escherichia coli populations have evolved over 3 y in a spatially heterogeneous and nutritionally complex environment. Metagenomic sequencing revealed that these initially isogenic populations evolved and maintained stable subpopulation structure in just 10 mL of medium for up to 10,000 generations, consisting of up to five major haplotypes with many minor haplotypes. We characterized the genomic, transcriptomic, exometabolomic, and phenotypic differences between clonal isolates, revealing subpopulation structure driven primarily by spatial segregation followed by differential utilization of nutrients. In addition to genes regulating the import and catabolism of nutrients, major polymorphisms of note included insertion elements transposing into fimE (regulator of the type I fimbriae) and upstream of hns (global regulator of environmental-change and stress-response genes), both known to regulate biofilm formation. Interestingly, these genes have also been identified as critical to colonization in uropathogenic E. coli infections. Our findings illustrate the complexity that can arise and persist even in small cultures, raising the possibility that infections may often be promoted by an evolving and complex pathogen population.

Limited Mutation-Rate Variation Within the Paramecium aurelia Species Complex.

Mutation is one of the most fundamental evolutionary forces. Studying variation in the mutation rate within and among closely-related species can help reveal mechanisms of genome divergence, but such variation is unstudied in the vast majority of organisms. Previous studies on ciliated protozoa have found extremely low mutation rates. In this study, using mutation-accumulation techniques combined with deep whole-genome sequencing, we explore the germline base-substitution mutation-rate variation of three cryptic species in the Paramecium aurelia species complex-P. biaurelia, P. sexaurelia, and P. tetraurelia We find that there is extremely limited variation of the mutation rate and spectrum in the three species and confirm the extremely low mutation rate of ciliates.

Insights into an Extensively Fragmented Eukaryotic Genome: De Novo Genome Sequencing of the Multinuclear Ciliate Uroleptopsis citrina.

Ciliated protists are a large group of single-celled eukaryotes with separate germline and somatic nuclei in each cell. The somatic genome is developed from the zygotic nucleus through a series of chromosomal rearrangements, including fragmentation, DNA elimination, de novo telomere addition, and DNA amplification. This unique feature makes them perfect models for research in genome biology and evolution. However, genomic research of ciliates has been limited to a few species, owing to problems with DNA contamination and obstacles in cultivation. Here, we introduce a method combining telomere-primer PCR amplification and high-throughput sequencing, which can reduce DNA contamination and obtain genomic data efficiently. Based on this method, we report a draft somatic genome of a multimacronuclear ciliate, Uroleptopsis citrina. 1) The telomeric sequence in U. citrina is confirmed to be C4A4C4A4C4 by directly blunt-end cloning. 2) Genomic analysis of the resulting chromosomes shows a "one-gene one-chromosome" pattern, with a small number of multiple-gene chromosomes. 3) Amino acid usage is analyzed, and reassignment of stop codons is confirmed. 4) Chromosomal analysis shows an obvious asymmetrical GC skew and high bias between A and T in the subtelomeric regions of the sense-strand, with the detection of an 11-bp high AT motif region in the 3' subtelomeric region. 5) The subtelomeric sequence also has an obvious 40 nt strand oscillation of nucleotide ratio. 6) In the 5' subtelomeric region of the coding strand, the distribution of potential TATA-box regions is illustrated, which accumulate between 30 and 50 nt. This work provides a valuable reference for genomic research and furthers our understanding of the dynamic nature of unicellular eukaryotic genomes.

Estimating Seven Coefficients of Pairwise Relatedness Using Population-Genomic Data.

Population structure can be described by genotypic-correlation coefficients between groups of individuals, the most basic of which are the pairwise relatedness coefficients between any two individuals. There are nine pairwise relatedness coefficients in the most general model, and we show that these can be reduced to seven coefficients for biallelic loci. Although all nine coefficients can be estimated from pedigrees, six coefficients have been beyond empirical reach. We provide a numerical optimization procedure that estimates all seven reduced coefficients from population-genomic data. Simulations show that the procedure is nearly unbiased, even at 3× coverage, and errors in five of the seven coefficients are statistically uncorrelated. The remaining two coefficients have a negative correlation of errors, but their sum provides an unbiased assessment of the overall correlation of heterozygosity between two individuals. Application of these new methods to four populations of the freshwater crustacean Daphnia pulex reveal the occurrence of half siblings in our samples, as well as a number of identical individuals that are likely obligately asexual clone mates. Statistically significant negative estimates of these pairwise relatedness coefficients, including inbreeding coefficients that were typically negative, underscore the difficulties that arise when interpreting genotypic correlations as estimations of the probability that alleles are identical by descent.

Population Genomics of Paramecium Species.

Population-genomic analyses are essential to understanding factors shaping genomic variation and lineage-specific sequence constraints. The dearth of such analyses for unicellular eukaryotes prompted us to assess genomic variation in Paramecium, one of the most well-studied ciliate genera. The Paramecium aurelia complex consists of ∼15 morphologically indistinguishable species that diverged subsequent to two rounds of whole-genome duplications (WGDs, as long as 320 MYA) and possess extremely streamlined genomes. We examine patterns of both nuclear and mitochondrial polymorphism, by sequencing whole genomes of 10-13 worldwide isolates of each of three species belonging to the P. aurelia complex: P. tetraurelia, P. biaurelia, P. sexaurelia, as well as two outgroup species that do not share the WGDs: P. caudatum and P. multimicronucleatum. An apparent absence of global geographic population structure suggests continuous or recent dispersal of Paramecium over long distances. Intergenic regions are highly constrained relative to coding sequences, especially in P. caudatum and P. multimicronucleatum that have shorter intergenic distances. Sequence diversity and divergence are reduced up to ∼100-150 bp both upstream and downstream of genes, suggesting strong constraints imposed by the presence of densely packed regulatory modules. In addition, comparison of sequence variation at non-synonymous and synonymous sites suggests similar recent selective pressures on paralogs within and orthologs across the deeply diverging species. This study presents the first genome-wide population-genomic analysis in ciliates and provides a valuable resource for future studies in evolutionary and functional genetics in Paramecium.

Diversity and Universality of Endosymbiotic Rickettsia in the Fish Parasite Ichthyophthirius multifiliis.

Although the presence of endosymbiotic rickettsial bacteria, specifically Candidatus Megaira, has been reported in diverse habitats and a wide range of eukaryotic hosts, it remains unclear how broadly Ca. Megaira are distributed in a single host species. In this study we seek to address whether Ca. Megaira are present in most, if not all isolates, of the parasitic ciliate Ichthyophthirius multifiliis. Conserved regions of bacterial 16S rRNA genes were either PCR amplified, or assembled from deep sequencing data, from 18 isolates/populations of I. multifiliis sampled worldwide (Brazil, Taiwan, and USA). We found that rickettsial rRNA sequences belonging to three out of four Ca. Megaira subclades could be consistently detected in all I. multifiliis samples. I. multifiliis collected from local fish farms tend to be inhabited by the same subclade of Ca. Megaira, whereas those derived from pet fish are often inhabited by more than one subclade of Ca. Megaira. Distributions of Ca. Megaira in I. multifiliis thus better reflect the travel history, but not the phylogeny, of I. multifiliis. In summary, our results suggest that I. multifiliis may be dependent on this endosymbiotic relationship, and the association between Ca. Megaira and I. multifiliis is more diverse than previously thought.

Disentangling the Taxonomy of Rickettsiales and Description of Two Novel Symbionts ("Candidatus Bealeia paramacronuclearis" and "Candidatus Fokinia cryptica") Sharing the Cytoplasm of the Ciliate Protist Paramecium biaurelia.

In the past 10 years, the number of endosymbionts described within the bacterial order Rickettsiales has constantly grown. Since 2006, 18 novel Rickettsiales genera inhabiting protists, such as ciliates and amoebae, have been described. In this work, we characterize two novel bacterial endosymbionts from Paramecium collected near Bloomington, IN. Both endosymbiotic species inhabit the cytoplasm of the same host. The Gram-negative bacterium "Candidatus Bealeia paramacronuclearis" occurs in clumps and is frequently associated with the host macronucleus. With its electron-dense cytoplasm and a distinct halo surrounding the cell, it is easily distinguishable from the second smaller symbiont, "Candidatus Fokinia cryptica," whose cytoplasm is electron lucid, lacks a halo, and is always surrounded by a symbiontophorous vacuole. For molecular characterization, the small-subunit rRNA genes were sequenced and used for taxonomic assignment as well as the design of species-specific oligonucleotide probes. Phylogenetic analyses revealed that "Candidatus Bealeia paramacronuclearis" clusters with the so-called "basal" Rickettsiales, and "Candidatus Fokinia cryptica" belongs to "Candidatus Midichloriaceae." We obtained tree topologies showing a separation of Rickettsiales into at least two groups: one represented by the families Rickettsiaceae, Anaplasmataceae, and "Candidatus Midichloriaceae" (RAM clade), and the other represented by "basal Rickettsiales," including "Candidatus Bealeia paramacronuclearis." Therefore, and in accordance with recent publications, we propose to limit the order Rickettsiales to the RAM clade and to raise "basal Rickettsiales" to an independent order, Holosporales ord. nov., inside Alphaproteobacteria, which presently includes four family-level clades. Additionally, we define the family "Candidatus Hepatincolaceae" and redefine the family Holosporaceae IMPORTANCE: In this paper, we provide the characterization of two novel bacterial symbionts inhabiting the same Paramecium host (Ciliophora, Alveolata). Both symbionts belong to "traditional" Rickettsiales, one representing a new species of the genus "Candidatus Fokinia" ("Candidatus Midichloriaceae"), and the other representing a new genus of a "basal" Rickettsiales According to newly characterized sequences and to a critical revision of recent literature, we propose a taxonomic reorganization of "traditional" Rickettsiales that we split into two orders: Rickettsiales sensu stricto and Holosporales ord. nov. This work represents a critical revision, including new records of a group of symbionts frequently occurring in protists and whose biodiversity is still largely underestimated.

Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli.

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10(-4) insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10(-5) recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186 Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5'-GGGG(N6/N7)CCCC-3'. We also detected 48 long deletions not involving IS elements.

Genomic and Metagenomic Analysis of Diversity-Generating Retroelements Associated with Treponema denticola.

Diversity-generating retroelements (DGRs) are genetic cassettes that can produce massive protein sequence variation in prokaryotes. Presumably DGRs confer selective advantages to their hosts (bacteria or viruses) by generating variants of target genes-typically resulting in target proteins with altered ligand-binding specificity-through a specialized error-prone reverse transcription process. The only extensively studied DGR system is from the Bordetella phage BPP-1, although DGRs are predicted to exist in other species. Using bioinformatics analysis, we discovered that the DGR system associated with the Treponema denticola species (a human oral-associated periopathogen) is dynamic (with gains/losses of the system found in the isolates) and diverse (with multiple types found in isolated genomes and the human microbiota). The T. denticola DGR is found in only nine of the 17 sequenced T. denticola strains. Analysis of the DGR-associated template regions and reverse transcriptase gene sequences revealed two types of DGR systems in T. denticola: the ATCC35405-type shared by seven isolates including ATCC35405; and the SP32-type shared by two isolates (SP32 and SP33), suggesting multiple DGR acquisitions. We detected additional variants of the T. denticola DGR systems in the human microbiomes, and found that the SP32-type DGR is more abundant than the ATCC35405-type in the healthy human oral microbiome, although the latter is found in more sequenced isolates. This is the first comprehensive study to characterize the DGRs associated with T. denticola in individual genomes as well as human microbiomes, demonstrating the importance of utilizing both individual genomes and metagenomes for characterizing the elements, and for analyzing their diversity and distribution in human populations.

The Rate and Spectrum of Spontaneous Mutations in Mycobacterium smegmatis, a Bacterium Naturally Devoid of the Postreplicative Mismatch Repair Pathway.

Mycobacterium smegmatis is a bacterium that is naturally devoid of known postreplicative DNA mismatch repair (MMR) homologs, mutS and mutL, providing an opportunity to investigate how the mutation rate and spectrum has evolved in the absence of a highly conserved primary repair pathway. Mutation accumulation experiments of M. smegmatis yielded a base-substitution mutation rate of 5.27 × 10(-10) per site per generation, or 0.0036 per genome per generation, which is surprisingly similar to the mutation rate in MMR-functional unicellular organisms. Transitions were found more frequently than transversions, with the A:T→G:C transition rate significantly higher than the G:C→A:T transition rate, opposite to what is observed in most studied bacteria. We also found that the transition-mutation rate of M. smegmatis is significantly lower than that of other naturally MMR-devoid or MMR-knockout organisms. Two possible candidates that could be responsible for maintaining high DNA fidelity in this MMR-deficient organism are the ancestral-like DNA polymerase DnaE1, which contains a highly efficient DNA proofreading histidinol phosphatase (PHP) domain, and/or the existence of a uracil-DNA glycosylase B (UdgB) homolog that might protect the GC-rich M. smegmatis genome against DNA damage arising from oxidation or deamination. Our results suggest that M. smegmatis has a noncanonical Dam (DNA adenine methylase) methylation system, with target motifs differing from those previously reported. The mutation features of M. smegmatis provide further evidence that genomes harbor alternative routes for improving replication fidelity, even in the absence of major repair pathways.

Draft Genome Sequence of Caedibacter varicaedens, a Kappa Killer Endosymbiont Bacterium of the Ciliate Paramecium biaurelia.

Caedibacter varicaedens is a kappa killer endosymbiont bacterium of the ciliate Paramecium biaurelia. Here, we present the draft genome sequence of C. varicaedens.