{'fraction_non_alphanumeric': 0.03806228373702422, 'fraction_numerical': 0.030084582852748943, 'mean_word_length': 5.086282538754022, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Comparison of Intracellular “Ca. Endomicrobium Trichonymphae” Genomovars Illuminates the Requirement and Decay of Defense Systems against Foreign DNA

“Candidatus Endomicrobium trichonymphae” (Bacteria; Elusimicrobia) is an obligate intracellular symbiont of the cellulolytic protist genus Trichonympha in the termite gut. A previous genome analysis of “Ca. Endomicrobium trichonymphae” phylotype Rs-D17 (genomovar Ri2008), obtained from a Trichonympha agilis cell in the gut of the termite Reticulitermes speratus, revealed that its genome is small (1.1 Mb) and contains many pseudogenes; it is in the course of reductive genome evolution. Here we report the complete genome sequence of another Rs-D17 genomovar, Ti2015, obtained from a different T. agilis cell present in an R. speratus gut. These two genomovars share most intact protein-coding genes and pseudogenes, showing 98.6% chromosome sequence similarity. However, characteristic differences were found in their defense systems, which comprised restriction-modification and CRISPR/Cas systems. The repertoire of intact restriction-modification systems differed between the genomovars, and two of the three CRISPR/Cas loci in genomovar Ri2008 are pseudogenized or missing in genomovar Ti2015. These results suggest relaxed selection pressure for maintaining these defense systems. Nevertheless, the remaining CRISPR/Cas system in each genomovar appears to be active; none of the “spacer” sequences (112 in Ri2008 and 128 in Ti2015) were shared whereas the “repeat” sequences were identical. Furthermore, we obtained draft genomes of three additional endosymbiotic Endomicrobium phylotypes from different host protist species, and discovered multiple, intact CRISPR/Cas systems in each genome. Collectively, unlike bacteriome endosymbionts in insects, the Endomicrobium endosymbionts of termite-gut protists appear to require defense against foreign DNA, although the required level of defense has likely been reduced during their intracellular lives.


Introduction
Many invertebrates and single-celled eukaryotes (protists) harbor intracellular symbionts, which generally contribute to the host's nutrition and/or defense systems (Moran et al. termites (Ohkuma and Kudo 1996;Hongoh et al. 2003Hongoh et al. , 2005Yang et al. 2005;Boucias et al. 2013;Dietrich et al. 2014;Abdul Rahman et al. 2015;Tai et al. 2015). The majority of Endomicrobium species are found as intracellular symbionts of various protist species in the termite gut Ikeda-Ohtsubo et al. 2007;Ohkuma et al. 2007). No freeliving phase has been observed for these endosymbionts, and the relationship with the protist host is strictly speciesspecific (Ikeda- Ohtsubo et al. 2007;Ohkuma et al. 2007;. A sole cultured representative of this genus is a free-living species, Endomicrobium proavitum, which has been isolated from a termite gut (Zheng, Dietrich, Radek, et al. 2016). No endosymbiotic Endomicrobium species has been cultivated thus far.
"Candidatus Endomicrobium trichonymphae" is an intracellular symbiont of the protist genus Trichonympha (phylum Parabasalia) Ohkuma et al. 2007). Trichonympha species are widespread in the gut of lower termites and play a pivotal role in cellulose digestion (Yamin 1979(Yamin , 1981. Previous phylogenetic analysis showed that Trichonympha species and their Endomicrobium endosymbionts have strictly cospeciated, and it was estimated that their symbiosis commenced 73-38 million years ago (Ikeda- Ohtsubo and Brune 2009). Among the 16S rRNA phylotypes of "Ca. Endomicrobium trichonymphae", phylotype Rs-D17 specifically inhabits the cytoplasm of Trichonympha agilis in the gut of the termite Reticulitermes speratus (Hongoh et al. 2003;Ohkuma et al. 2007). The complete genome sequence of phylotype Rs-D17 was previously obtained using a whole genome amplification (WGA) technique from a single host T. agilis cell (Hongoh et al. 2008a). The genome analysis suggested that the endosymbiont imports glucose 6-phosphate as a carbon and energy source, which is expected to be abundant in the cellulolytic host cytoplasm. Phylotype Rs-D17, in turn, likely provides amino acids and cofactors, which are deficient in dead wood, to their host protists and termites. The Rs-D17 genome is small (1.1 Mb) and corresponds to about two-thirds of the genome size of the free-living isolate Endomicrobium proavitum (1.6 Mb) (Hongoh et al. 2008a;. In addition, the Rs-D17 genome contains 121 pseudogenes, which account for approximately 15% of the predicted protein-coding sequences (CDSs) (Hongoh et al. 2008a). These suggest that phylotype Rs-D17 is in the course of reductive genome evolution, as seen in many other intracellular bacterial symbionts (McCutcheon and Moran 2012).
In the present study, we reconstructed the complete genome sequence of another genomovar, designated as Ti2015, of phylotype Rs-D17 from a different T. agilis cell present in an R. speratus worker's gut. To discriminate the two genomovars, we designated the Rs-D17 genome obtained in the previous study (Hongoh et al. 2008a) as genomovar Ri2008. By comparing these two closely related genomovars, we aimed to elucidate the evolutionary process of the endosymbionts within a short time scale.

Comparison of General Features of Genomovars Ti2015 and Ri2008
The Ti2015 genome comprised a circular chromosome and three circular plasmids (table 1), which showed very high sequence similarities to the respective genome components of genomovar Ri2008 ( fig. 1 and supplementary fig. S1, Supplementary Material online). The two genomovars had identical 16S rRNA gene sequences and shared 97.0% sequence identity in the internal transcribed spacer (ITS) regions of the rRNA operon. The overall chromosome nucleotide sequence identity was 98.6%, and the nucleotide sequence identity in the CDSs was 99.6% on average. The chromosome size of Ti2015 is 11.3 kb smaller than that of Ri2008 (table 1). This difference in genome size is largely attributed to indels longer than 1 kb that are distributed around the entire genomes ( fig. 1). The number of intact CDSs on the chromosome of Ti2015 was lower (719 vs. 761) and the number of pseudogenes was higher (150 vs. 121) (table 1). All of the 719 CDSs in Ti2015 are present in Ri2008. The frequency of small indels < 3 bp in the pseudogenes was as high as that in the intergenic regions (supplementary table S1  between closely related bacterial strains, although the mechanism is unknown (Eisen et al. 2000;Tillier and Collins 2000).
Recently, it has been suggested that restriction-modification (R-M) systems might have caused the massive genome rearrangements present between "Ca. Endomicrobium trichonymphae" and Endomicrobium proavitum (Zheng, Dietrich, Hongoh, et al. 2016). We could not find evidence for involvement of R-M systems in this large inversion or other rearrangements between the Ti2015 and Ri2008 genomes. Most of the genes and pseudogenes were shared by the two genomovars, including the pseudogenes of the chromosome replication initiator protein DnaA, ribosomal protein S1 (RpsA) and glutamine synthetase (GlnA) (supplementary table S2, Supplementary Material online) (Hongoh et al. 2008a). On the other hand, clear differences were found in the defense systems comprising restriction-modification systems and clustered regularly interspaced short palindromic repeat (CRISPR) regions with its associated genes (cas genes).

Decay and Conservation of Defense Systems
Genomovars Ti2015 and Ri2008 possess three or two intact R-M systems, respectively, in addition to numerous . For example, a type II R-M system (RSTT_354 and RSTT_355) of genomovar Ti2015 is pseudogenized in genomovar Ri2008, where the restriction endonuclease gene was split by the large inversion mentioned above ( fig. 1). This gene corruption likely allowed the pseudogenization of the adjacent DNA methyltransferase (RSTT_355) in Ri2008. Likewise, many R-M system-related genes are pseudogenized in only one of the two genomovars (table 2), whereas most other intact CDSs and pseudogenes are shared. This suggests that the decay of R-M systems has occurred randomly and has proceeded rapidly, compared with other genes.
The CRISPR/Cas system is an adaptive and heritable defense mechanism: foreign DNA fragmentized by the action of Cas proteins are recorded as "spacers" flanked by "repeat" sequences in a CRISPR region (Karginov and Hannon 2010). Genomovar Ri2008 has three CRISPR/Cas systems ( fig. 2). According to the classification proposed by Makarova et al. (2011), one system belongs to type I-C and the other two belong to type II-C. The two sets of the type II-C cas genes are phylogenetically distinct and designated here as type II-C-a FIG. 1.-Synteny of chromosomes between genomovars Ri2008 and Ti2015. Upper and lower columns indicate the chromosomes of genomovars Ri2008 and Ti2015, respectively. Red lines show the regions with 90% nucleotide sequence identity between the genomovars. Yellow wedges indicate the positions of deletions 1kb length. A schematic view of the pseudogenization of an R-M system caused by a large inversion is also shown. A restriction endonuclease gene in Ti2015 (RSTT_354: orange box) was split into two parts by the inversion and found as pseudogene fragments in Ri2008 (two dashed orange boxes). An adjacent methyltransferase gene (RSTT_355: green box) was also split into two and found as pseudogene fragments in Ri2008 (two dashed green boxes). The length of the boxes indicates the relative sequence length. Wavy ends of CDSs indicate split sites. Other genes around the inverted region are not shown, and omitted regions are indicated by double slashes. and b, respectively, for convenience ( fig. 3 and supplementary  fig. S4, Supplementary Material online). Genomovar Ti2015 retained the type I-C system, but the type II-C-a is pseudogenized and the type II-C-b is completely missing (fig. 2). The cas9 gene of the type II-C-a in Ri2008 has been split into two parts, which probably caused the loss of the adjacent CRISPR region as in Ti2015. Taken together with the ongoing decay of the R-M systems, these results suggest that the defense systems of these bacteria have been under relaxed selection pressure, likely because of their intracellular lifestyle, which affords protection within host eukaryotic cells.
Nevertheless, none of the 112 and 128 "spacer" sequences of the type I-C CRISPR/Cas system in Ri2008 and Ti2015, respectively, was shared, whereas the "repeat" sequences are identical (supplementary table S3, Supplementary Material online). This indicates that at least the type I-C CRISPR/Cas system is actively working in "Ca. Endomicrobium trichonymphae" phylotype Rs-D17. Indeed, transcripts of the cas7/csd2 and cas1 genes of the type I-C system were detected by the reverse transcription polymerase chain reaction (RT-PCR) (data not shown). Thus, the "Ca. Endomicrobium trichonymphae" phylotype Rs-D17 appears to have been subjected to invading foreign DNA even after taking up residence inside T. agilis cells.

R-M and CRISPR/Cas Systems in Other Endomicrobium Endosymbionts
To examine whether a genome with intact CRISPR/Cas systems and numerous intact or pseudogenized R-M systems is unique to phylotype Rs-D17 or common in Endomicrobium endosymbionts, we additionally reconstructed draft genomes of three endosymbiotic Endomicrobium phylotypes associated with different host protist species: phylotype HsTcC-EM16 from Trichonympha sp. HsjTcC in the gut of the termite Hodotermopsis sjostedti; phylotype MdDo-005 from the protist Deltotrichonympha operculata and phylotype MdMp-027 from the protist Mixotricha paradoxa in the gut of the termite Mastotermes darwiniensis. These three Endomicrobium phylotypes indeed possess numerous R-M systems including many pseudogenes (supplementary table S4, Supplementary Material online) and also apparently intact CRISPR/Cas systems ( fig. 2). Many of the R-M systems are shared by two or more of the endosymbiotic phylotypes, whereas the free-living isolates in the phylum Elusimicrobia, Elusimicrobium minutum, and Endomicrobium proavitum, possess only few R-M systems (supplementary table S4, Supplementary Material online) (Herlemann et al. 2009;Zheng, Dietrich, Hongoh, et al. 2016). It is therefore likely that most of the numerous R-M systems were acquired by common ancestors of the endosymbionts after divergence from the Endomicrobium proavitum lineage, as suggested previously (Zheng, Dietrich, Hongoh, et al. 2016). Although the lifestyles of those ancestors are unknown, they likely needed an array of defense systems for protection against phages and other invading DNA elements.
Endosymbiotic and free-living Endomicrobium species commonly have an intact type I-C CRISPR/Cas system ( fig. 2), and each of the cas1, cas2, and cas4 genes of the Endomicrobium species constituted a monophyletic cluster ( fig. 4a and supplementary fig. S5a,b, Supplementary Material online). However, the remaining gene components, cas3, cas5, cas7/csd2, and The type II-C-a system is commonly found in Endomicrobium species, although the system appears to have lost its function in phylotype Rs-D17 as discussed above ( fig. 2), and the system is absent from Endomicrobium proavitum. Each of the cas genes of this system in the Elusimicrobia formed a monophyletic cluster ( fig. 3, and supplementary fig. S4a,b, Supplementary Material online), and the tree topology is congruent with that of the rRNA gene-based tree (supplementary fig. S6, Supplementary Material online). The type II-C-b system in Ri2008 was found also in phylotype HsTcC-EM16, although the CRISPR region of the latter is missing. Phylotype HsTcC-EM16 possesses another phylogenetically distinct gene set of the type II-C system, designated here as II-C-c, accompanying two separate CRISPR regions, which have identical "repeat" sequences. The type-II-C-c system was also found in Endomicrobium proavitum (fig. 2). These results indicate that at least the type I-C and type II-C-a systems of the endosymbiotic Endomicrobium phylotypes have been inherited from a common ancestor, even though the former system experienced the replacement of a part of the cas gene cluster.

Requirement of Defense against Foreign DNA in Intracellular Symbionts
Most reported intracellular symbionts of insects are housed in specialized host cells called bacteriocytes, allowing sequestration away from the insect body or gut lumen. The necessity of the defense systems against foreign DNA for these organisms is therefore expected to be low. Endosymbiotic Endomicrobium species also inhabit the cytoplasm of eukaryotic cells; however, their protist hosts are exposed to the gut luminal fluid that contains a dense community of prokaryotes and presumably phages. Because the protist hosts phagocytose wood particles, other gut luminal components such as smaller protists, prokaryotic cells, phages, and extracellular nucleic acids could simultaneously be endocytosed (Yamaoka and Nagatani 1977). It is conceivable that the endosymbionts are exposed to phages and DNA released from digested microbial cells, as discussed previously (Zheng, Dietrich, Hongoh, et al 2016), even though the frequency of exposure is likely to be much lower than for gut bacteria living outside the protist cells (i.e., in the gut lumen). Thus, it might be expected that endosymbiotic Endomicrobium species require defense systems against foreign DNA. On the other hand, "Candidatus Azobacteroides pseudotrichonymphae" (phylum Bacteroidetes; order Bacteroidales), an intracellular symbiont of the protist Pseudotrichonympha grassii in the gut of the termite Coptotermes formosanus, has neither R-M systems nor CRISPR/Cas systems (Hongoh et al. 2008b), despite its genome size and the number of CDSs being comparable to "Ca. Endomicrobium trichonymphae". Thus, the requirement of such defense systems may also depend on the bacterial taxonomic groups and/or other unknown factors.

Sample Collection
The termite R. speratus was collected at Ogose in Saitama Prefecture, Japan in 2012. This is the same site where the R. speratus individual used for reconstructing the Ri2008 genome was collected in 2006. H. sjostedti was collected in Kagoshima Prefecture, Japan, and M. darwiniensis was collected in Darwin, Australia. Worker termites reared with cellulose powder for 3 days were subjected to the experiments. Single-cell isolation of gut protists and collection of their endosymbiotic Endomicrobium cells were performed as described previously (Hongoh et al. 2008a), with an addition of 0.1% Tween 20 (Sigma-Aldrich) (Sato et al. 2014). WGA was performed with the illustra GenomiPhi HY DNA Amplification Kit (GE Healthcare Life Sciences) for 8 h, as described previously (Hongoh et al. 2008b).
Although we did not analyze the phylogenetic relationship between the T. agilis cells that hosted the Ti2015 and Ri2008 genomovars, there has been no report that there are multiple phylotypes of T. agilis in R. speratus guts. Considering the very high genome sequence identity of the endosymbionts Ti2015 and Ri2008 genomovars and the strict host-specificity of Endomicrobium endosymbionts , the host T. agilis cells were most likely highly similar.

Genome Sequencing and Bioinformatics
Libraries for paired-end and mate-pair sequencing were prepared using the TruSeq DNA PCR-free Sample Prep Kit and the Nextera Mate Pair Sample Prep Kit (Illumina), respectively. Sequencing was performed on an Illumina MiSeq platform using the MiSeq Reagent Kit v3 (600 cycles). The generated reads were quality-filtered and assembled into contigs using SPAdes 3.1 (Bankevich et al. 2012), which were further assembled to scaffolds using SCARPA 0.241 (Donmez and Brudno 2013). Gaps in scaffolds were closed by PCR amplification and Sanger sequencing on ABI3730 Genetic Analyzers. Detailed procedures are described in the supplementary methods (Supplementary Material online). CDSs were predicted using the BLASTn algorithm on the basis of the annotation for genomovar Ri2008 (Hongoh et al. 2008a). The sequence alignment with genomovar Ri2008 was manually checked. Pseudogenes were identified as described previously (Hongoh et al. 2008a), and single nucleotide polymorphisms were counted manually. Synteny of the genome regions was analyzed using GenomeMatcher 2.0 (Ohtsubo et al. 2008). Chromosome sequence identity was calculated using Genome-to-Genome Distance Calculator 2.1 (Meier-Kolthoff et al. 2013). The dif sequence was predicted on the basis of the deduced amino acid sequence of the site-specific recombinase XerCD, using a prediction system in the web site "Database of bacterial replication terminus" (http://www.glanguage.org/data/repter/) (Kono et al. 2012). The "repeat" and "spacer" sequences of CRISPRs were identified using CRISPRFinder (Grissa et al. 2007). The cas genes and R-M system-related genes in the draft genomes of the Endomicrobium phylotypes were identified by BLASTx searches against the NCBI non-redundant protein sequence database with default settings.

Phylogenetic Analysis
Nucleotide or deduced amino acid sequences were aligned using the ClustalW algorithm implemented in MEGA6.0 (Tamura et al. 2013). The best-fit substitution models were selected using Model test and used for constructing maximum-likelihood trees in MEGA6.0. For bootstrap analysis, 1,000 resamplings were performed. Bayesian trees were constructed using MrBayes 3.2.5 (Ronquist et al. 2012). Four Markov chains (three heated and one cold) were run simultaneously for 1,000,000 generations.

RNA Extraction and RT-PCR
Gut contents of 10 R. speratus workers were suspended in the Trager U solution (Trager 1934) in a 1.5 ml tube. The suspension was centrifuged with a low speed for collection of large protists including T. agilis, and the precipitate was resuspended in sterilized double-distilled water. Total RNA was extracted using the PowerViral Õ Environmental RNA/DNA Isolation Kit (MO BIO Laboratories). The TURBO DNA-free TM Kit (Ambion) was used to remove DNA. Reverse transcription was performed with random hexamers using the SuperScript TM III First-Strand Synthesis System for RT-PCR (Invitrogen). Primers specific to each of cas1 and csd2 genes were designed (supplementary table S5, Supplementary Material online). RT-PCR was performed using Ex-Taq polymerase (TaKaRa) on a C1000 Thermal Cycler (Bio-Rad). The following program was used: 3-min initial denaturation at 95 C, 30 cycles of denaturation (15 s at 95 C), annealing (30 s at 60 C), extension (30 s at 72 C), and a final 3-min extension at 72 C. No specific amplification was detected from the RNA samples without reverse transcription. The amplicons were directly sequenced with the same primers on an ABI3730 Genetic Analyzer.