Comprehensive evolutionary analysis of re- verse ...Comprehensive evolutionary analysis of reverse transcriptases in viruses and prokaryotes 5 “RNase_H_like Superfamily”. In many

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Report for Taikichiro Mori Memorial Research Grants 2019 (2019 年度森基金研究成果報告書)

生命の複製に関わる酵素の新規発見と機能解明 Comprehensive evolutionary analysis of re-verse transcriptases in viruses and prokary-otes Shohei Nagata Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0035, Japan and Sys-tems Biology Program, Graduate School of Media and Governance, Keio University, Fu-jisawa 252-0882, Japan.

Abstract Reverse transcriptases (RTs) are enzymes that polymerize DNA from RNA tem-plates. RTs are usually thought to be viral and eukaryotic elements, but they are also present in bacteria. Bacterial RTs are seemed to be ancestors of eukaryotic RTs and several types are identified i.e. group II introns, retrons, CRISPR/Cas-associated RTs, diversity-generating retroelements (DGRs), and Abi -like genes. Recently, several studies reported that the existence of RTs in a recently reported bacterial group, candidate phyla radiation (CPR). These CPR RTs are thought to have an important role and functions in CPR bacterial ecologies since they retain RT genes while lacking numerous biosynthetic pathways. In this study, I compre-hensively collected RT-like sequences from CPR genomes and systematically char-acterized RT functions and evolution. Using known functional domain profiles in RTs as queries, sequence similarity search was performed against 804 near-complete genomes of CPR bacteria in the database. I obtained 514 RT sequences and these RTs are widely distributed in CPR phyla. It is known that CPR bacteria utilize RTs involved in DGRs to adapt rapidly changing environments, I found RTs related to group II introns, retrons, and abortive infection (Abi). I will discuss possible roles and evolution of RTs in CPR bacteria. Contact: [email protected]

1 Introduction Central dogma in molecular biology is a flow of in-formation that genetic information retained on DNA is transcribed into mRNA and translated into protein, which was proposed in 1958. However, in 1970, an RNA-dependent DNA polymerase (reverse tran-scriptase; RT), which synthesizes DNA based on RNA, reversed this flow [1,2]. This was discovered by studies of tumor-associated retroviruses that in-fect eukaryotes, and various types of RT enzymes have been discovered primarily related to eukary-

otes thereafter. In addition to viruses infecting eu-karyotic organisms (retrovirus, pararetrovirus, hepadnavirus), the existence of a RT homologous region in long terminal repeat (LTR) retroelement, non-LTR retroelement, telomerase has been re-vealed.

In 1989, retron, one of the reverse transcriptase (RT) was found in bacteria [3,4]. Even after that, various types of RTs were discovered in bacteria and archaea by the discovery of group II intron [5–7] and diversity-generating retroelements (DGRs) [8–10] etc. Retrons consist of an RT and an adjacent repeat sequence but its function remains unknown.

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Group II introns are retroelements consists of cata-lytic RNA and an RT protein which mediate splic-ing and mobility reactions [11–13]. DGRs are retro-elements that lost mobility functions and use reverse transcription to generate sequence variations in spe-cific target genes [10]. Then, it was revealed that RT is a gene that is widely present in the three domains of life (bacteria, archaea, eukaryotes) and viruses [14–17]. In bacteria, it is also known that RT ho-mologous region exists also in abi gene related to abortive infection (Abi) to phage [18,19] and cas1 gene of CRISPR/Cas immune system [20,21]. Three bacterial RT-related proteins are involved in phage resistance; AbiA, AbiK, and Abi-P2 [15]. AbiA and AbiK are thought to provide phage im-munity through abortive infection. Also, recently there have been reports that many uncharacterized RT-like sequences mainly exist in bacteria [15,20,21]. However, what kind of functions/activi-ties they possess, and how they divergences were unclear.

More recently, it has become clear that a vast un-known microbial strain group exists in bacteria by technological advances in metagenomic analysis and single-cell genomics. Metagenomic approach revealed huge diversity of previously unknown phyla of bacteria and archaea since they have differ-ent forms of 16S rRNA sequences. In bacteria, these metagenomically recovered bacterial strain was de-scribed as candidate phyla radiation (CPR) and comprises at least 15% of all bacteria [22]. The CPR seems to be monophyletic and clearly separated from other bacteria (Figure 1.1; Castelle and Banfield, 2018; Hug et al., 2016). CPR bacteria are widely distributed across the various environments such as human microbiome [25] , deep subsurface sediments [26], the dolphin mouth [27], drinking water [28], soil [29], marine sediment [30] and other environments [24,31].

CPR bacteria have various unusual features com-pared to non-CPR bacteria. CPR genomes are less than 1.5Mb while the genome size of non-CPR bac-teria, Escherichia coli, is 4.6Mb. Most of them lost TCA-cycle genes and they have intron regions in rRNA genes [22,31]. It is sometimes questioned whether CPR bacteria is a cellular organism, at least, CPR genomes encode genetic systems for cell divi-sion (e.g. Fts-Z-based mechanisms, not found in some symbionts with very reduced genomes), and measurements of replication rates and images show-ing cell division indicate that the cells are metabol-ically active. It is also thought that they may adhere to the surface of other microorganisms to survive.

It is reported that CPR bacteria have RT-like se-quences in their genomes, however, the types of RTs, their functions, and its evolutionary scenario

of diversification are not well understood. In this study, a comprehensive analysis was performed on the RT sequence from CPR bacterial genomes, to reveal roles and evolution of RTs in CPR bacteria.

Figure 1.1 A current view of the tree of life. The phy-logenetic tree of bacteria, archaea, and eukaryotes, in-cluding 92 named bacterial phyla, 26 archaeal phyla and all five of the Eukaryotic supergroups. The tree was esti-mated by maximum-likelihood method using concatena-tion of ribosomal protein sequences. The figure adapted from reference [23].

2 Methods

2.1 Data sources Complete genome sequences of bacteria and ar-chaea were downloaded from the Reference Se-quence Database (RefSeq) [32] at the National Cen-ter for Biotechnology Information (NCBI) as of May 2018. The acquired genomes (denoted as Ref-Seq prokaryotes in this manuscript) were 9,078 ge-nomes (total of bacteria 8,825, archaea 253, respec-tively).

Nearly full-length (restored by ≥ 70% based on the estimated full length) of 804 genomes (790 spe-cies) of CPR bacteria were obtained from NCBI GenBank based on Hug et al. [23].

Known RT sequences were obtained from a pre-vious study by Simon et al. [20]. Sequences anno-tated as “Unknown”, “Unclassified”, and “nonRTs” were eliminated and totally 930 RT sequences were collected.

0.4

Candidate Phyla Radiation

Microgenomates

Parcubacteria

Eukaryotes

Archaea

Bacteria

DPANN

Opisthokonta

AmoebozoaChromalveolata

ArchaeplastidaExcavata

RBX1WOR1

Cyanobacteria

Melainabacteria

PVC superphylum

TACK

Major lineage lacking isolated representative: Major lineages with isolated representative: italics

Dojkabacteria WS6

PeregrinibacteriaGracilibacteria BD1-5, GN02

Absconditabacteria SR1

Katanobacteria WWE3

Berkelbacteria

SM2F11

CPR1CPR3

Nomurabacteria KaiserbacteriaAdlerbacteria

Campbellbacteria

Wirthbacteria

Chloroflexi

Armatimonadetes

GiovannonibacteriaWolfebacteria

Jorgensenbacteria

Azambacteria

YanofskybacteriaMoranbacteria

MagasanikbacteriaUhrbacteria

Falkowbacteria

Saccharibacteria

Woesebacteria

AmesbacteriaShapirobacteria

CollierbacteriaPacebacteria

BeckwithbacteriaRoizmanbacteria

GottesmanbacteriaLevybacteria

DaviesbacteriaCurtissbacteria

NanoarchaeotaWoesearchaeota

Pacearchaeota

Nanohaloarchaeota

Micrarchaeota

Altiarchaeales

Aenigmarchaeota

Diapherotrites

Z7ME43

Loki.

Thaumarchaeota

ArchaeoglobiMethanomicrobia

Halobacteria

Thermoplasmata

Methanococci

Spirochaetes

Firmicutes

(Tenericutes)

BacteroidetesChlorobi

Gammaproteobacteria

Alphaproteobacteria

Betaproteobacteria

Actinobacteria

PlanctomycetesChlamydiae,Lentisphaerae,Verrucomicrobia

Omnitrophica

Aminicentantes Rokubacteria

NC10

Elusimicrobia

Poribacteria

Ignavibacteria

Dadabacteria

TM6

Atribacteria

Gemmatimonadetes

CloacimonetesFibrobacteres

Nitrospirae

Latescibacteria

TA06

Caldithrix

Marinimicrobia

WOR-3

Zixibacteria

SynergistetesFusobacteria

AquificaeCalescamantes

Deinococcus-Therm.

CaldisericaDictyoglomi

Deltaprotebacteria(Thermodesulfobacteria)

Epsilonproteobacteria

DeferribacteresChrysiogenetes

Tectomicrobia, ModulibacteriaNitrospinae

Acidobacteria

Zetaproteo.

Thermotogae

Acidithiobacillia

Parvarchaeota

Hydrogenedentes NKB19

Thor.

BRC1

ThermococciMethanobacteria

Hadesarchaea

Methanopyri

Aigarch.

Crenarch.

YNPFFA

Korarch.

Bathyarc.

Figure 1 | A current view of the tree of life, encompassing the total diversity represented by sequenced genomes. The tree includes 92 named bacterialphyla, 26 archaeal phyla and all !ve of the Eukaryotic supergroups. Major lineages are assigned arbitrary colours and named, with well-characterized lineagenames, in italics. Lineages lacking an isolated representative are highlighted with non-italicized names and red dots. For details on taxon sampling and treeinference, see Methods. The names Tenericutes and Thermodesulfobacteria are bracketed to indicate that these lineages branch within the Firmicutes andthe Deltaproteobacteria, respectively. Eukaryotic supergroups are noted, but not otherwise delineated due to the low resolution of these lineages. The CPRphyla are assigned a single colour as they are composed entirely of organisms without isolated representatives, and are still in the process of de!nition atlower taxonomic levels. The complete ribosomal protein tree is available in rectangular format with full bootstrap values as Supplementary Fig. 1 and inNewick format in Supplementary Dataset 2.

LETTERS NATURE MICROBIOLOGY DOI: 10.1038/NMICROBIOL.2016.48

NATURE MICROBIOLOGY | www.nature.com/naturemicrobiology2

© 2016 Macmillan Publishers Limited. All rights reserved

Comprehensive evolutionary analysis of reverse transcriptases in viruses and prokaryotes

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2.2 Identification of RT sequences From the prokaryotic genomes collected, RT-like sequences which have RT functional domains were identified using HMMER v.3.2 (hmmscan program; E-value ≤ 1e-5) [33] search against sequence pro-files corresponding to “RVT_1” (PF00078) or “RVT_2” (PF07727) in Pfam-A 32.0 [34]. In our first pipeline, Pfam ID: “RVT_3” (PF13456) was included in the query profile since “RVT_3” do-main was registered as “Reverse transcriptase-like” in the database. However, proteins collected with “RVT_3” profile query were RNase H protein ra-ther than RT. Therefore, I exclude proteins exist as RNase H alone, not a part of RT protein, to observe the diversity and evolution of RT domains and pro-teins in the analysis including CPR bacteria.

2.3 Network analysis based on sequence sim-

ilarities The sequence similarity scores were calculated to construct a weighted undirected graph (SSN). The similarity scores (Basic Local Alignment Search Tool [BLAST] bit scores) [35] for all the collected protein sequences were calculated with an all-against-all BLASTP (BLAST 2.7.1+) analysis [36,37], with a cut-off E-value of ≤ 1e−5. Using the BLAST bit scores, the sequence similarities were normalized to 0.0–1.0, with the following equation [38,39]:

𝒔𝒊𝒎(𝒙, 𝒚) =𝐦𝐚𝐱(𝒃𝒊𝒕𝒔𝒄𝒐𝒓𝒆(𝒙, 𝒚), 𝒃𝒊𝒕𝒔𝒄𝒐𝒓𝒆(𝒚, 𝒙))𝐦𝐚𝐱(𝒃𝒊𝒕𝒔𝒄𝒐𝒓𝒆(𝒙, 𝒙), 𝒃𝒊𝒕𝒔𝒄𝒐𝒓𝒆(𝒚, 𝒚))

where sim(x,y) represents the normalized sequence similarity between two sequences x and y. If the score was 1.0, the pair was deemed to be identical. A weighted undirected graph was constructed based on the scores of all the pairs of sequences, and the edges were weighted with the scores. I set a thresh-old sequence identity value and connected the nodes when the sequence identity exceeded the threshold. The threshold to be used was determined by com-paring the networks constructed with an incremen-tal series of threshold values. The constructed net-works were visualized with Cytoscape 3.7.1 [40], using “Prefuse Force-Directed OpenCL Layout” with default parameters except for enabling “Force deterministic layouts” option.

2.4 Sequence comparison and phylogenetic

analysis To compare differences between RefSeq prokary-otic RT and CPR bacteria RT, I aligned RT se-

quences using MAFFT v.7.407 (L-INS-i algo-rithms) [41] and estimated maximum likelihood tree using RAxML v.8.2.11 [42] with PROTGAMMAJTT evolutionary model for amino acid sequences. Both analyses were performed and visualized through the environment for tree explo-ration (ETE) v.3.1.1 [43]. Also, the identified CPR RTs were mapped onto the phylogenetic tree esti-mated by Hug et al. [23] using iTOL [44].

2.5 Estimation of frameshift mutations in

polymerases To verify whether the frameshift mutation occurred only in CPR bacterial RTs, DNA polymerase family A proteins were identified and compared to the RTs. DNA polymerase family A proteins were identified using HMMER v.3.2 (hmmscan program; E-value ≤ 1e-5) [33] search against sequence profiles corre-sponding to “DNA_pol_A” (PF00476) in Pfam 32.0 [34]. To increase phylogenetic coverage of the pol-ymerases in CPR phylogeny, the retrieved DNA polymerase protein sequences (438 sequences for CPR bacteria) were additionally run against all cod-ing sequences of datasets using BLAST v.2.8.1+ (blastp program; E-value ≤ 1e-5; query coverage per subject ≥ 50%) [35–37] and 670 sequences were identified for CPR bacteria. With the same pipeline, I also re-identified RT sequences using 514 CPR RT sequences as query and retrieved 539 RTs from CPR genomes.

2.6 Domain architecture of related proteins Domain organization of CPR RTs were visualized with DoMosaics v.0.95 [45]. The visualized do-mains were extracted using HMMER v.3.2 (hmm-scan program) [33] search against Pfam-A 32.0 [34] database. HMMER was performed and the results were combined by DoMosaics. Other sequences which have specific domain architecture was searched by InterProScan [46] against InterPro da-tabase [47].

2.7 Identification of RT-related group II in-

trons Since most of bacterial group II introns have RT as intron-encoded protein (IEP) in its open reading frame (ORF), I identified the introns to annotate RT functions. To detect its characteristic RNA second-ary structures surrounding IEP (RT), homologous structures to the specific domains of the introns (do-mains I-VI) in CPR bacterial genomes were searched. Domains V, VI were searched using In-

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fernal v.1.1.2 (cmsearch program with --nohmm op-tion; score > 24) against RNA secondary structural profiles corresponding to “Intron_gpII” (RF00029) in Rfam database [48]. For domains I-IV, Infernal v.1.1.2 (cmsearch program with --rfam option; E-value ≤ 1e-10) were used against profiles corre-sponding to “group-II-D1D4-1” (RF01998), “group-II-D1D4-2” (RF01999), “group-II-D1D4-3” (RF02001), “group-II-D1D4-4” (RF02003), “group-II-D1D4-5” (RF02004), “group-II-D1D4-6” (RF02005), and “group-II-D1D4-7” (RF02012) in the database. Based on the search results, consider-ing the distances between the intron components, types of group II introns were defined as follows; full-length, which has all domains I-IV, ORF-RT, domains V-VI; ORF-less, which lacks ORF-RT but has domains; others which lacks one of the three components.

3 Results and discussion

2.1 Overall relationships among prokaryot-

ic RTs To see overall sequence relationship of RT and RT-related proteins in prokaryotes and viruses, I con-structed and visualized sequences sequence similar-ity network (SSN) (Figure 3.1). The SSN is a graphical representation of the similarities between sequences. Each sequence is indicated by a point (node) and the similarity between the sequences is represented by the length of the line (edge) connect-ing the points. The smaller the distance between the nodes, the greater the degree of similarity between the sequences. I used RT and the related protein se-quences identified from prokaryotic and viral ge-nomes in RefSeq dataset. Nodes are colored accord-ing to the origin of sequences: bacteria (non-CPR); archaea; virus (Figure 3.1A) or to the types of RT and RT-related proteins (Figure 3.1B). An over-view of the entire network structure shows that the RT and RT-related proteins can be divided into four groups, i.e., RTs of bacteria and archaea, RTs of vi-ruses, RNA-dependent RNA polymerases (RdRp), Ribonuclease (RNase) H. The group of viral RTs and viral RdRp consisted only of sequences derived from viruses, whereas the group of RNase H and bacterial RT both contained sequences derived from thee domains. Some bacterial type of RTs, such as DGR, have been found in virus (bacteriophage) ge-nomes [49], and they are mainly associated with the bacterial RT group on the network.

The phylogenetic relationships of the obtained RTs and RT-related proteins were analyzed together with structure of protein functional domains

(Figure 3.2). Several sequences were selected from each type of RT and used. The color of tips in the tree corresponds to the color of the node in Figure 3.1, and the type of RT and the taxonomic domain (bacteria, archaea, virus) derived from are described together. Retroviral, LTR, non-LTR, and retron II types of RTs were located nearby on the phyloge-netic tree, while group II introns and retron I RTs were splitted and located on multiple strains. Many RTs of the virus possessed various protein domains in addition to the central domain of the RT, as de-scribed “RVT_1” in the figure, and the sequence length was considerably longer than that of prokar-yotes. This is probably because viruses often encode one protein with multiple functions.

Figure 3.1 Sequence similarity network of RTs from RefSeq prokaryotes. Nodes (colored dots) represent the RT protein sequences and the edge lengths represent the sequence similarities. (A) Nodes are colored according to the origin of sequences: bacteria (non-CPR); archaea; vi-rus. (B) Nodes are colored according to the types of RT and RT-related proteins.

RdRp and RNase H, which is not RT itself, were obtained as RT-related proteins. RdRp has been considered to be evolutionary related to RT [50] and it is not surprising that the RdRp domain sequences were highly similar to the RT domain. On the other hand, for RNase H, I selected Pfam ID: “RVT_3” in the process of selecting protein sequences having the RT domain. Although the “RVT_3” domain are registered as “Reverse transcriptase-like” in NCBI CDD, the superfamily does not belong to “RVT_1 Superfamily” and “RT_like Superfamily” with the other RTs but the superfamily belong to

BacteriaArchaeaVirus

RNase H

RNA dependent RNA polymerase

RT ZFREV-like

RT Rtv

RT LTR

Viral DNA polymerase

RT retronⅠ

RT nLTR-like

RT group II intron RT retronⅡ

RdRP 3

RdRP 4

Unclassified

A

B


5

“RNase_H_like Superfamily”. In many cases, RT has an RNase H domain region as part of it [51,52]. However, after this, I exclude proteins exist as RNase H alone, not a part of RT protein, to observe the diversity and evolution of RT domains and pro-teins in the subsequent analysis including CPR bac-teria.

To analyze the characteristics of RT in CPR bac-teria, I firstly plotted histograms of amino acid se-quence length of RTs extracted from CPR bacteria and non-CPR prokaryotes registered in RefSeq (Figure 3.3). Only when plotting histograms, Ref-Seq prokaryotic RTs were used for cluster repre-sentative sequences to which at least 5 sequences belong to each cluster in order to ensure sequence

Figure 3.2 Phylogenetic tree and domain architecture of RTs. Based on the RT and RT-related proteins identi-fied in the RefSeq prokaryotic genomes, several se-quences were obtained from each type of RT. The color-ing of the tip of the phylogenetic tree corresponds to the coloring of the node in Figure 3.1. Also, the type of RT and the taxonomic domain (bacteria, archaea, virus) de-rived from were described. Functional protein domains are colored by domain type and names of domain in Pfam databases are indicated.

diversity. Sequence length of each RT dataset shows that the minimum length was 78 residues for CPR bacteria and 72 residues for RefSeq prokary-otes, the mean length was 311 residues and 475 res-idues, and the maximum length was 763 residues and 1879 residues respectively. The shape of the distribution showed that the RT of CPR bacteria was unimodal and had a small variation in sequence length, while the RefSeq prokaryotes had roughly three peaks with a multimodal distribution. As a re-sult, the RT of the prokaryote registered in RefSeq contains a wide variety of RT types, whereas most of the RT of CPR bacteria are specific types of RT.

For comparing sequence between RTs in CPR bacteria and non-CPR prokaryotes, I constructed and visualized SSN of RTs from both datasets (Figure 3.4). Note that in Figure 3.1, Pfam ID: “RVT_3” was also included in the extraction of RTs. However, a considerable number of RNase H se-quences were included in the network. These RNase H protein profile (Pfam ID: “RVT_3”) was ex-cluded since I would like to target only sequences close to the RT enzyme. CPR bacteria RTs, which nodes are colored blue, showed a cluster-like se-quences on the left side of the network and se-quences scattered slightly to the lower left

Figure 3.3 Distribution of sequence length of the iden-tified RT proteins. Distribution of amino acid sequence length of the identified RTs from (A) CPR RT (B) non-CPR prokaryotic RTs registered in RefSeq database. Note that panel B is a representative sequence of clusters containing 5 or more sequences due to reduce the bias in the sequence data of RefSeq.

A

B

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Figure 3.4 Sequence similarity network of RTs from RefSeq (non-CPR) prokaryotes and CPR bacteria. Nodes (colored dots) represent the RT protein sequences and the edge lengths represent the sequence similarities. (A) Nodes are colored according to the origin of se-quences: bacteria (non-CPR); CPR bacteria; archaea; vi-rus. (B) Nodes are colored according to the types of RT and RT-related proteins.

(Figure 3.4A). These RTs were classified as group II intron type and retron type, respectively (Figure 3.4B). In addition to these, some CPR bacterial RTs have been annotated as RNase H-like proteins (5 se-quences) or seemed to be similar to viral RdRp (3 sequences).

Nodes annotated as group II introns type of RT from CPR bacteria were clustered on the network (Figure 3.4) and seemed to be consists majority of the CPR RTs. Previous study reported that 75% of the RT in the bacterial genome belongs to the group II intron, with 12% for the retron and 3% for the DGR [15,53]. However, it should be noted that, as mentioned above, a detailed discussion must be made in conjunction with a more accurate RT type annotation. This cluster of sequences is might be characteristic RTs of CPR bacteria because of its distance compared to bacteria and archaeal RT other than CPR bacteria on the network. If these were RT associated with Group II introns as noted, new types of Group II introns might be present in CPR bacteria. Since this RT annotation was determined only by

the best hits in the NCBI CDD profiles, detailed types would be identified by phylogenetic analysis with known types of RTs in the next section.

2.2 Functional analysis and classification of

CPR RTs Sequence similarity-based search of RT domains identified 514 RT protein sequences. To observe the phylogenetic distribution of the RTs, they were mapped onto CPR bacterial phylogenies [23] (Figure 3.5). RTs were widely distributed in CPR bacteria. They appeared in both major superphyla of CPR, Parcubacteria (OD1) and Microgenomates. RTs were found in 313 species out of 804 of CPR bacteria.

I combined CPR RT sequences and the known RT sequences and constructed phylogenetic tree (Figure 3.6). The CPR RTs were not monophyletic, and RTs related to retrons, abortive infection (AbiK, Abi-P2, but not AbiA), DGRs, group II introns and group II intron-like were observed in CPR. Most of CPR RTs (441 sequences) were involved in DGRs and it consists 86% of CPR RTs

Figure 3.5 Phylogenetic distribution of RTs in CPR bacteria. RTs were found in 313 genomes and they were mapped onto the CPR phylogeny (804 genomes). Ge-nomes with RT proteins are colored in blue. The CPR phylogeny was taken from Hug et al. and modified.

CPR BacteriaArchaeaVirus

Bacteria

RNA-dependent RNA polymerase

RNase H-like

RT ZFREV-like

RT Rtv

RT LTR

Viral DNA polymerase

RT retronⅠ

RT nLTR-like

RT group II intron

RT retronⅡ

RdRP 3

RdRP 4

Others & Unclassified

Cas1

A

B


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Figure 3.6 Phylogenetic relationships of CPR RTs and known RTs. 514 sequences of CPR RTs and 930 se-quences of known RTs were combined, and the phyloge-netic tree were estimated using maximum-likelihood method. The types of RTs are indicated at right side of the tree nodes (tips). Collapsed nodes are of known RT sequences except for 416 CPR RT sequences indicated as star.

Most CPR RTs have only RT domain, but five RT sequences have other domains (Figure 3.8). Three of them (GenBank accession, KKR03365.1; KKU32234.1; OGV95898.1) have “GIIM” (Pfam ID, PF08388) domain which is maturase-specificdomain of group II intron. Also, protein OGV95898.1 has “RVT_N” (PF13655) domain which means N-terminal domain of reverse tran-scriptase. Interestingly, the other two proteins have specific domains, “zf-CHC2” (PF01807) and “UDG”

(PF03167). “zf-CHC2” is a domain of CHC2-type zinc finger domain which bind metals such as zinc, iron, or no metal at all. “UDG” is a domain of uracil DNA glycosylase (UDG). UDG is an enzyme that reverts mutations in DNA and crucial in DNA repair. I further searched sequences by domain architecture which have both RT domain and UDG domain against the protein database of almost all public available sequences. However, the only sequences I found was sequences of Candidatus Giovannoni-bacteria, which host species is same as the sequence I mentioned (OGF82770.1). Therefore, I concluded that the RT which have UDG is specific in Candi-datus Giovannonibacteria.

To observe RTs in bacterial group II introns, the introns were detected by searching their character-istic RNA secondary structures (Table 3.1). Totally 12 group II introns were detected and six of them had RTs as IEP. The remaining six group II introns had both domains I-IV and domains V-VI, but the distance between the domains were short (~450 nt) that they didn’t possess corresponding RTs.

The phylogenetic mapping and the aforemen-tioned analysis revealed several types of RT pro-teins were widely distributed and conserved in CPR bacterial phyla. The roles and functions of RTs in CPR bacterial ecologies were less understood ex-cept for DGRs [16,54]. DGRs, retroelements that generates sequence variations in specific target genes using RT, were identified in CPR bacteria and suggested to be utilized for adaptation to a dy-namic host-dependent environment [54]. The ob-served phylogenetic distribution of CPR RTs was congruent with the previous study which identified DGRs [54]. Despite the study was focused on RTs as component of DGRs, I newly identified RTs in CPR phyla, i.e. Candidatus Collierbacteria, Candi-datus Pacebacteria, subdivision RIF-10, 15, 16, 17, 20, 21 of Candidatus Parcubacteria. Especially, RTs in RIF-10, 15, 16, 17, 20, 21 of Parcubacteria were identified in multiple genomes that indicates non-DGR RTs exist in the phyla and may play important role for their ecologies.

The identified group II introns were seemed to be recently acquired by horizontal gene transfer since they have well conserved group II intron maturase domain and their RTs are closely related to RTs

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2 @149195205 01872295.1

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C01000036.1@C 2011 C2 48 [email protected]@@ @1619071@3198 4356

��

………………………………………☆

Retrons

Abi-like

DGRs

Group II introns

Table 1. Identified group II introns in CPR bacteria

Genomes (GenBank accession) Exsistence of RNA domains and RTCorresponding RTs(GenBank accession)

Microgenomates_group_bacterium_RBG_16_45_19 (MHDC01000039.1) Full (Domain I-IV, ORF-RT, Domain V,VI) OGV95898.1

Candidatus_Kerfeldbacteria_bacterium_RIFCSPLOWO2_02_FULL_42_19 (MHKF01000008.1) Full (Domain I-IV, ORF-RT, Domain V,VI) OGY84268.1

Candidatus_Uhrbacteria_bacterium_GW2011_GWF2_46_218 (LCMG01000021.1) Full (Domain I-IV, ORF-RT, Domain V,VI) KKU32234.1

Candidatus_Uhrbacteria_bacterium_GW2011_GWF2_39_13 (LBWG01000031.1) ORF-RT, Domain V,VI KKR03365.1

Candidatus_Vogelbacteria_bacterium_RIFOXYB1_FULL_42_16 (MHTH01000005.1) Domain I-IV, ORF-RT OHA59186.1

Candidate_division_Kazan_bacterium_RIFCSPLOWO2_01_FULL_48_13 (METE01000004.1) Domain I-IV, ORF-RT OGB85388.1

Candidatus_Levybacteria_bacterium_RIFCSPHIGHO2_01_FULL_37_33 (MFNM01000037.1) ORF-less (Domain I-IV, Domain V,VI)

Candidatus_Levybacteria_bacterium_RIFCSPLOWO2_02_FULL_37_10 (MFPB01000045.1) ORF-less (Domain I-IV, Domain V,VI)

Candidatus_Colwellbacteria_bacterium_RIFCSPHIGHO2_12_FULL_44_17 (MHIX01000008.1) ORF-less (Domain I-IV, Domain V,VI)

Candidatus_Nealsonbacteria_bacterium_RBG_13_42_11 (MHLY01000006.1) ORF-less (Domain I-IV, Domain V,VI)

Candidatus_Terrybacteria_bacterium_RIFCSPLOWO2_01_FULL_40_23 (MHSW01000005.1) ORF-less (Domain I-IV, Domain V,VI)

Candidatus_Gottesmanbacteria_bacterium_GW2011_GWB1_49_7 (LCQD01000034.1) ORF-less (Domain I-IV, Domain V,VI)

Table 3.1 Identified group II introns in CPR bacteria.

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from non-CPR prokaryotes. RTs involved in abor-tive infection and retron were also identified. Abor-tive infection is a process which provide phage im-munity through blocking phage multiplication by programmed death of the cell. Three bacterial RT-related proteins are involved in phage resistance; AbiA, AbiK, and Abi-P2. AbiK and Abi-P2 were identified in CPR bacterial genome in this study, and several RTs that closed to AbiK but formed dis-tinct clade were also identified. Also, retrons consist of an RT gene and an adjacent inverted repeat se-quence, but its function remains unknown. Detailed sequence properties of these RTs will be investi-gated for further research.

Despite the CPR bacteria phyla were radiation and clearly separated from the other bacteria, the RT tree were polyphyletic. This result implies that CPR bacteria and the other non-CPR bacteria RTs ex-change RT genes occasionally, contributing RT evolution and diversifications. I identified several types of RTs and biological properties of these pro-teins were not elucidated, but the RTs might con-tribute to CPR ecology since the RTs still exist and not discarded in extremely small CPR genomes

Figure 3.8 Domain architecture of CPR RTs with other domains. Functional domains in RT proteins are visualized. Domains are based on Pfam database and de-fined as follows: RVT_1 (PF00078), Reverse transcrip-tase (RNA-dependent DNA polymerase); GIIM (PF08388), Group II intron, maturase-specific domain; RVT_N (PF13655), N-terminal domain of reverse tran-scriptase; UDG (PF03167), Uracil DNA glycosylase su-perfamily; zf0CHC2 (PF01807), CHC2 zinc finger.

2.3 Sequence analysis of small CPR RTs im-

plies putative ribosomal frameshifts To observe features of CPR RT, I compare se-quences of CPR RT and usual bacterial RT (Figure 3.7). Compared to group II intron RT (in the intron-encoded protein), retron, and DGR RT, multiple Se-quence alignments showed that some CPR RTs have very short sequences and it seemed to be trun-cated since their first-half regions were well aligned but they didn’t have latter half regions.

I hypothesized that these truncations in coding se-quences were occurred by ribosomal frameshift so that the latter half regions may exist in the down-stream region of the coding region. To confirm the hypothesis, the downstream regions were concate-nated with their coding sequences and aligned (Figure 3.9). The downstream regions of those trun-cated RTs were well aligned to full-length (not trun-cated) RT, so it seemed that frameshift mutation had occurred at the end of the coding region.

Figure 3.9 Sequence alignment of RT-coding regions and its neighbors. Representative truncated RTs and their downstream regions were concatenated and aligned with a full-length (not truncated) RT (protein ID: KKS64476.1 of Parcubacteria group bacterium GW2011 GWB1 42 6). Black dashed box represents putative translational frameshift site.

LBWG01000031.1@[email protected]@@RNA-directed_DNA_polymerase__Reverse_transcriptase_@taxon|1618995@8899_9832 RVT_1 GIIM

LCMG01000021.1@[email protected]@@Reverse_transcriptase__RNA-dependent_DNA_polymerase_@taxon|1619001@2782_4459 RVT_N RVT_1 GIIM

MFIA01000018.1@Candidatus_Giovannonibacteria_bacterium_RIFCSPLOWO2_01_FULL_44_16@OGF82770.1@@hypothetical_protein@taxon|1798348@11033_12213 RVT_1 UDG

MFLC01000025.1@Candidatus_Kaiserbacteria_bacterium_RIFCSPHIGHO2_02_FULL_49_11@OGG55025.1@@hypothetical_protein@taxon|1798489@58351_59635 RVT_1 zf-CHC2

MHDC01000039.1@[email protected]@@group_II_intron_reverse_transcriptase/maturase@taxon|1817747@2779_4027 RVT_1 GIIM

Domain architecture

KKR03365.1

KKU32234.1

OGF82770.1

OGG55025.1

OGV95898.1

Protein (GenBank accession)

Full-length RT

RT① CDS CDS + downstream

RT② CDS CDS + downstream

RT③ CDS CDS + downstream

RT④ CDS CDS + downstream

RT⑤ CDS CDS + downstream

Full-length RT






Full-length RT






Full-length RT






Fig. 2.

CPR RT

Fig. 1.

Fig. 1.

Figure 3.7 Phylogenetic relationships and schematic sequence alignment of RT protein sequences. Six CPR RTs and three the other RTs (RT of group II introns, DGRs, retrons, respectively) were aligned and schematically represented in the right side. Grey colored box represents the existence of residues and the other regions are gaps of alignments. CPR RTs are surrounded with a blue square.


9

To observe the phylogenetic distribution of these putative frameshift RTs, the RTs were mapped onto CPR bacterial phylogenies [23] (Figure 3.10A). Pu-tative frameshift RTs were not inclined to appear in specific phyla, but they were widely distributed in CPR bacteria. They appeared in both major super-phyla of CPR, Parcubacteria (OD1) and Microge-nomates. RTs were found in 318 species out of 804 of CPR bacteria and putative frameshift RTs existed in 38 species. Totally, 63 out of 539 RTs (11.7%) and 367 out of 7,143 RTs (5.1%) were identified as frameshift RT in CPR bacteria and RefSeq prokar-yotes, respectively.

To confirm that the frameshift occurred especially in RT, DNA polymerase family A proteins were re-trieved and analyzed frameshift likewise (Figure 3.10B). In contrast to RT, only 4 out of 670 DNA polymerase family A proteins (0.6%) were identi-fied as frameshift protein and they were found in only 4 species.

From this analysis, I found that RT proteins con-taining frameshift mutations were widely conserved in CPR bacteria phyla. It was difficult to think that these proteins were conserved without any reasons, and so I assumed that the proteins might be fully translated with translational frameshift, the reading ribosomes slipped and skip nucleotides and read a different frame hereafter.

There were few studies that comprehensively identified RTs in CPR bacteria, except for DGRs [16,54]. DGRs, retroelements consists of RT and specific types of related sequences, were identified in CPR bacteria and suggested to be utilized for ad-aptation to a dynamic host-dependent environment [54]. The phylogenetic distribution of CPR RTs was congruent with the previous study which identified DGRs [54]. Despite the study was focused on DGR RTs, I newly identified RTs in CPR phyla, i.e. Can-didatus Amesbacteria, Candidatus Collierbacteria, Candidatus Pacebacteria. The types of RTs which I identified were not clearly identified and the biolog-ical properties of these phyla were not elucidated, but the RTs might contribute to CPR ecology since the RTs still exist and not discarded in extremely small CPR genomes.

Considering that putative translational frameshifts existed in RT, not other polymerases, some specific mechanisms related to RT might contribute to arise frameshift in RT. I supposed that RTs in bacterial group II introns contributed to the mutation. Group II introns are a kind of mobile elements and its mo-bility reactions are mediated by RTs encoded in the introns [6,7]. RTs synthesized DNA from tran-scribed group II introns, and frameshift mutation may arise during the reverse transcription. Despite

RTs in group II introns has high processive and fi-delity to preserving functions in the RT [12,55,56], RTs lack the 3’ to 5’ proofreading capability of other DNA polymerases [57] and prone to errors.

Since most of CPR bacteria genomes were con-structed by metagenomics, it was possible that RT protein frameshift was just an artifact of sequencing. Therefore, I observed protein frameshift in another protein, DNA polymerase protein, to reduce the possibility, and the result showed that protein frameshifts were only found in few cases implying that the frameshifts could not be an artifact of met-agenome methodologies.

Figure 3.10 Phylogenetic distribution of putative frameshift proteins. The existence of putative frameshift/full-length RTs (A) and DNA polymerase family A (B) proteins were mapped onto the CPR phy-logeny (804 genomes). Genomes with full-length pro-teins are colored in blue and putative frameshift proteins are colored in red. RTs were found in 318 species and putative frameshift RTs existed in 38 species, and DNA polymerase family A proteins were found in 619 species and the putative frameshift proteins existed in 4 species. The CPR phylogeny was taken from Hug et al. and mod-ified.

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4 Conclusion In this study, I revealed that several types of RT pro-teins were widely distributed and conserved in CPR bacterial phyla. At least, CPR bacteria have RTs re-lated to DGRs, group II introns, retrons, and abor-tive infection (Abi), and the abundance was differ-ent from non-CPR bacteria. While CPR bacteria thought to attach to the other micro-organism to live, the result of the majority of DGRs, which are minor in other prokaryotes, suggests that CPR bacteria have successfully utilized the property of DGRs, in-troducing mutations in the target genes, to adapt rapidly changing host environments. The polyphy-letic tree of CPR RTs implying that CPR bacteria and the other non-CPR bacteria RTs exchange genes occasionally, contributing RT evolution and diversifications. Also, Sequence comparisons among CPR RTs, the other prokaryotic and viral RTs showed that there were several truncated RT protein sequences. These were RTs containing frameshift mutations and widely distributed in CPR phyla. Since this phenomenon is RT-specific and it is unlikely that group II introns introducing muta-tions when replicating their own sequences, it is speculated that these RTs with frameshift mutations may retain some kind of functions.

Acknowledgements I would like to thank Professor Akio Kanai for his great support of my research. He taught me the fas-cinating aspects of molecular biology and evolution, and he always encouraging my research. I also thank Ms. Megumi Tsurumaki, Mr. Masahiro Miura, and all the members of the RNA Group for their in-sightful discussions. I would also like to express my gratitude to my family for their moral support and warm encouragement. Finally, I would like to thank Professor Masaru Tomita for providing a stimulat-ing environment to do my research in IAB. This work was supported, in part, by Taikichiro Mori Memorial Research Grants.

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