Detection of bacterial gene expression elements on Tobacco mosaic virus RNA using cDNA analysis

Yoshiyuki Nishimiya

doi:10.1371/journal.pone.0358128

Abstract

Tobacco mosaic virus (TMV) is a positive-stranded RNA virus that infects plants. Interestingly, the 5’-untranslated region (UTR) of the TMV RNA genome is recognized and translated by the ribosomes of Escherichia coli in a Shine–Dalgarno (SD) sequence-independent manner. This study aimed at investigation of the bacterial recognition modules that control gene expression within the TMV RNA genome. To this end, the 5’-end-complete cDNA of the TMV RNA and several 5’-end-truncated cDNA mutants, in which the movement protein-encoding gene and its downstream region were replaced with a DNA sequence encoding a green fluorescent protein, i.e., monomeric Umikinoko-Green (mUkG1), were constructed. Surprisingly, mUkG1 fluorescence was observed in E. coli transformants harboring the cloned cDNAs, although they were inserted into a vector lacking a promoter. Analysis of the 5’-end-truncated cDNA mutants and promoter prediction suggested that an E. coli-specific promoter might be located 2.1 kb upstream of the initiation codon for mUkG1. Furthermore, Western blotting analysis and conversion of the initiation codon ATG to AGT indicated that the translation of mUkG1 started from the correct initiation codon. These results imply that E. coli ribosomes correctly recognize the initiation codon on the mRNA, irrespective of the overly long 5’-UTR. To the best of our knowledge, this report is the first to reveal a recognizable bacterial module hidden within the TMV RNA genome through cDNA construction.

Citation: Nishimiya Y (2026) Detection of bacterial gene expression elements on Tobacco mosaic virus RNA using cDNA analysis. PLoS One 21(9): e0358128. https://doi.org/10.1371/journal.pone.0358128

Editor: Robert A. Niederman, Rutgers University/New Brunswick, UNITED STATES OF AMERICA

Received: February 27, 2026; Accepted: August 27, 2026; Published: September 15, 2026

Copyright: © 2026 Yoshiyuki Nishimiya. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This study was supported by JSPS KAKENHI Grant Number JP16K14909 (https://www.jsps.go.jp/). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Viruses lack a protein translation machinery and instead utilize their hosts’ cellular machinery to translate their mRNA and produce viral proteins, including structural capsid proteins and nonstructural proteins, such as replicases and RNA-silencing suppressor proteins. Viral genomes are composed of either DNA or RNA in the double-stranded (ds) or single-stranded (ss) forms [1]. The virome of eukaryotes mostly contains RNA viruses, especially positive-stranded RNA (+RNA) viruses [2]. In eukaryotic cells, the genetic material is enclosed within the nuclear envelope. Therefore, viruses infecting the cytoplasm of host cells cannot readily access the host’s DNA replication and transcription machinery [3,4]. Negative-stranded RNA (−RNA) and dsRNA viruses produce mRNAs in the host cytoplasm using their RNA genomes as templates, thereby initiating viral replication. Therefore, these viruses package their transcription and replication machineries into their virions [1,5,6]. Conversely, the genomes of +RNA viruses can function directly as mRNAs in the host cytoplasm, which might explain why RNA viruses, especially +RNA viruses, are predominant in the virome of eukaryotes [2].

Tobacco mosaic virus (TMV) is a + RNA virus belonging to the genus Tobamovirus and the family Virgaviridae, which can infect solanaceous plants. Its complete RNA genome sequence was reported in 1982 [7]. This genome encodes four viral proteins: a 126-kDa protein [comprising the methyltransferase (MT) domain, the intervening region (IR), and the RNA helicase (Hel) domains], the read-through 183-kDa replicases [comprising the 126-kDa protein and RNA-dependent RNA polymerase (RdRp) domains], the movement protein (MP), and the capsid protein (CP) [7–9]. The 3’-end of the gene encoding the 183-kDa protein serves as the subgenomic promoter (Sgp) and the N-terminal region for MP, while the 3’-end of the MP gene acts as the Sgp for CP [10]. The 5’-untranslated region (5’-UTR) of the TMV RNA genome, also known as the omega (Ω) sequence, contains a poly-CAA region, which functions as a translation enhancer in plants [11,12]. Conversely, the 3’-UTR can fold into a tRNA-like structure that contributes to efficient genome replication [13,14]. Interestingly, the TMV genome has been shown to replicate in non-native eukaryotic hosts, including Saccharomyces cerevisiae [15]. Moreover, reports have shown SD sequence-independent translation of the genomes of RNA viruses, including TMV, in bacteria, such as Escherichia coli [16,17]. The 5’-UTR sequences of TMV and Alfalfa mosaic virus RNA4 can be recognized with high specificity and translated by E. coli ribosomes. The ribosomal protein S1 in E. coli plays a critical role in ensuring accurate translation of these viral sequences [17,18]. While the heterologous translation in bacteria has been investigated, the genetic traces of gene expression elements in plant viral RNAs have remained largely unexplored.

Here, we generated the cDNA sequence of a modified TMV genome, comprising the 5’-UTR, the 183-kDa replicase-encoding region (including the Sgp for MP), and a monomeric Umikinoko-Green (mUkG1) gene (instead of the MP-encoding gene). The Sgp for CP, the CP-encoding gene, and the 3’-UTR were deleted. This cDNA sequence was cloned into a lac promoter-deleted pUC19 vector. Surprisingly, E. coli transformants harboring the constructs exhibited fluorescence, indicating that mUkG1 is expressed, despite the absence of a promoter and an SD sequence for the mUkG1 gene. In this study, to elucidate the elements that contribute to mUkG1 expression, a deletion analysis from the 5’-end of the modified TMV cDNA and promoter prediction were performed. These analyses suggest that the gene expression elements for mUkG1 are located in the TMV-Hel domain-encoding region, which is far away from the mUkG1-encoding DNA. Furthermore, a comparison with a highly homologous Tomato Mosaic virus (ToMV)-Hel indicated that a sequence resembling a bacterial promoter was located upstream of the gene encoding the RecA-like domain of TMV-Hel. These observations imply a genetic trace of gene expression elements in the TMV genome RNA, which might have been shared by an ancestor of +RNA viruses. Therefore, these findings may be significant for understanding the evolutionary scenarios for RNA viruses in the eukaryotic virome.

Materials and Methods

Recombinant strains and growth conditions used for vector construction and mUkG1 expression

The E. coli HST08 strain (TaKaRa Bio, Japan) was used for vector construction. The E. coli HST08 and KRX strains (Promega, USA) were used for analyzing the expression of mUkG1. E. coli cells were grown in Luria-Bertani (LB) medium containing 100 µg/mL ampicillin for pUC-TMx-G and 50 µg/mL kanamycin for pT7-TMx-G at 32 °C. For in vivo homologous recombination of DNA fragments, the Saccharomyces cerevisiae BY4743 (MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 LYS2/lys2Δ0 met15Δ0/MET15 ura3Δ0/ura3Δ0) strain (Open Biosystems, USA) was used. The S. cerevisiae cells harboring vectors containing the ura3 gene were grown in synthetic defined medium without uracil (Merck, Germany) at 30 °C.

Vector construction

All DNA fragments and PCR primers used are shown in S1 and S2 Tables. PCR amplifications were performed using the Q5 high-fidelity DNA polymerase (New England Biolabs, USA). Fourteen DNA fragments (TM1, TM2, TM3, TM4, TM5, TM6, TM7, TM8, TM9, TM10, TM11, TM12, TM13–14, and TM15) of the TMV gene and the mUkG1-encoding DNA were synthesized by AZENTA (Burlington, USA). To construct the cDNA for the modified TMV genome, overlap extension PCRs were first performed for each fragment pair (TM1/TM2, TM3/TM4, TM5/TM6, TM7/TM8, TM9/TM10, and TM11/TM12) using the respective primer set [TMx-F and TM(x + 1)-R], generating six extended fragments. These fragments, TM13–14 and TM15, along with the BamHI/XbaI-digested pCLY vector (ATTO, Japan), were introduced into S. cerevisiae BY4743 using the Frozen-EZ Yeast Transformation II Kit (ZYMO RESEARCH, USA). This resulted in the introduction of the modified TMV cDNA into pCLY via in vivo homologous recombination. The transformants were spread on agar plates containing synthetic defined medium without uracil and incubated for 2 days at 30 °C. The colonies formed were harvested and cultured in synthetic defined medium without uracil at 30 °C. The prepared constructs (denoted as pCLY-TM) were purified from the transformants using Zymoprep Yeast Plasmid Miniprep Kits (ZYMO RESEARCH, USA), amplified in the E. coli strain HST08, and recovered for subsequent vector construction. The pCLY-TM vector and the mUkG1-encoding DNA were digested with ZraI/AscI and AscI, respectively, and ligated (S1 Fig A). The ligated vector (denoted as pCLY-TM-G) was used as a PCR template to construct the deletion mutants from the 5’-end of the TMV gene and the promoter-less mUkG1 gene. Six PCR fragments were amplified using TM1-F, TM5-F, TM7-F, TM8-F, TM9-F, and mUkG1-F as forward primers and TM14-R as the reverse primer. These PCR fragments were digested with AvrII and then ligated to the SmaI/XbaI-digested pUC19 vector (denoted as pUC19-TMx-G and pUC19-G in S1 Fig B). To delete the lacZ promoter from pUC19, PCR was performed using pUC19-TMx-G and pUC19-G as the templates and the PvuII-forward/reverse primer set. The PCR amplicons were ligated to the PvuII-digested pUC19 vector using the In-Fusion cloning kit (TaKaRa Bio, Japan) (S1 Fig C). The details of the completed pUC-TMx-G vectors are provided in Fig 1. The pUC-TMx-G vectors were used as PCR templates to construct the T7 promoter-inducible vectors (pT7-TMx-G). Then, these PCR products were cloned into a vector based on the pET26b backbone. The promoter sequences are shown in S3 Table. The sequences of the generated TMV and mutant cDNAs were confirmed by Sanger sequencing.

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Fig 1. Construction of the pUC-TMx-G vectors.

The fragments containing the gene encoding the 5’-untranslated, methyltransferase, intervening, helicase, and RNA-dependent RNA polymerase (RdRp) regions of TMV and mUkG1 were cloned between the PvuII and PvuII sites of the pUC19 vector. The gene encoding RdRp contained the subgenomic promoter for the movement protein (MP), whose initiation codon was deleted via synonymous substitution. The bent arrow indicates the initiation codon for mUkG1.

https://doi.org/10.1371/journal.pone.0358128.g001

Expression analysis of mUkG1

Each E. coli HST08 colony harboring pUC-TM-G, its deletion mutant vectors, or pUC19 (as a negative control) was inoculated into 2 mL of LB medium containing ampicillin and pre-cultivated overnight at 32 °C. Then, 20 µL of the overnight culture was transferred into tubes containing 2 mL of freshly prepared LB medium with ampicillin and incubated at 145 rpm and 32 °C for 48 h. During the 48-h incubation, 100 µL of these cultures were transferred into each well of a 96-well microplate at different time points (4, 8, 12, 24, 36, and 48 h) to measure OD₆₀₀ and fluorescence intensity using a multimode microplate reader (Spark; TECAN, Switzerland). The fluorescence was normalized to the OD₆₀₀ values (fluorescence/OD₆₀₀). To estimate the background autofluorescence, the fluorescence of E. coli harboring pUC19 was also measured. For the KRX strain, 10 µL of the overnight culture was transferred into each well of a 96-well plate containing 1 mL of freshly prepared LB medium with kanamycin. The plates were incubated at 900 rpm and 32 °C. After 4 h, mRNA transcription was induced by adding 5 µL of 20% rhamnose for the T7 promoter. OD₆₀₀ and fluorescence intensity values were measured after an additional 6 h of culture. Furthermore, the E. coli cells harboring pUC-TMx-G were cultivated in 2 mL of LB medium for 24 h at 32 °C and collected by centrifugation for 5 min at 5,000 × g. The fluorescence of E. coli pellets was observed using a fluorescent excitation light-emitting diode (LED) source (VariRays I; ATTO, Japan).

The expression levels of mUkG1 were analysed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. For pUC-TM-G, pUC-TM5-G, pUC-TM7-G, and pUC-TM9-G, a C-terminal hexahistidine tag (6×His-tag) was conjugated to mUkG1 (denoted as pUC-TM-GH, pUC-TM5-GH, pUC-TM7-GH, and pUC-TM9-GH, respectively). After 24 h of culture, E. coli cell pellets harboring these vectors were lysed using the BugBuster Master Mix protein extraction reagent (Merck, USA). After normalizing total cellular protein content, the proteins were analysed together with a positive control, i.e., 6×-His-tagged mUkG1 prepared from the BL21 (DE3) strain. For Western blotting, the proteins separated by SDS-PAGE were transferred to polyvinylidene fluoride (PVDF) membranes and then detected using an anti-His-tag mouse monoclonal antibody (MBL, Japan) and a horseradish-peroxidase-conjugated anti-mouse IgG (H + L) goat polyclonal Fab (MBL, Japan) with Clarity Western ECL Substrate (BIO-RAD, USA).

Rapid amplification of cDNA 5’-end (5’-RACE)

To prepare total RNA, 270 µL of the E. coli culture, harvested after 24 h, was mixed with 540 µL of RNA Protect Bacteria Reagent (Qiagen, Germany). After incubation for 5 min, the cell suspensions were centrifuged for 10 min at 5,000 × g. Then, the pellets were resuspended in 200 µL of lysis buffer (30 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 15 mg/mL lysozyme) containing proteinase K. After incubation for 10 min, 700 µL of buffer RLT (Qiagen, Germany) containing 40 mM dithiothreitol (DTT) was added to the lysed pellets. Subsequently, total RNA was purified from these pellets using an RNeasy Mini kit (Qiagen, Germany) according to the manufacturer’s protocol. The prepared RNAs were treated with RNase-free DNase I (New England Biolabs, USA) for 1 h at 37 °C. Then, DNase I was heat-denatured for 10 min at 75 °C after the addition of 5mM EDTA. The DNase-treated RNAs were finally purified using the Monarch RNA Cleanup kit (New England Biolabs, USA).

5’-RACE was performed with 5’-Full RACE Core Set (TaKaRa Bio, Japan) according to the manufacturer’s protocol. First-strand cDNAs were synthesized using 2.5 µg of total RNA as the template, 200 pmol of 5’-end-phosphorylated primers for reverse transcription (S2 Table), and 5 U of AMV RT XL for 10 min at 30 °C. This was followed by elongation for 60 min at 50 °C and denaturation for 2 min at 80 °C. Then, the hybrid RNA was degraded using RNase H for 60 min at 30 °C. The single-stranded cDNA was collected from the mixture by ethanol precipitation. Next, these single-stranded cDNAs were circularized or concatenated using T4 RNA ligase for 18 h at 15 °C.

The first PCR was performed using KOD FX Neo (TOYOBO, Japan) with the 1^st PCR primer sets and a 10-fold dilution of circularized cDNAs or concatemers as templates. The conditions for the 1^st PCR were as follows: one cycle of denaturation for 2 min at 94 °C, 30 cycles of denaturation for 10 s at 98 °C, annealing for 30 s at 65 °C, and elongation for 1 min at 68 °C. The 1^st PCR products were diluted 10 or 100-fold and then used as templates for nested PCR (2^nd PCR). The conditions for the nested PCR using Taq polymerase (New England Biolabs, USA) and 2^nd PCR primer sets were as follows: one cycle of denaturation for 30 s at 95 °C, and 30 cycles of denaturation for 30 s at 95 °C, annealing for 15 s at 49 °C, and elongation for 2 min at 68 °C. The 2^nd PCR products were TA-cloned into the pMD19 vector (TaKaRa Bio, Japan). The 5’-ends of cloned cDNAs were determined by Sanger sequencing using BigDye Terminator Cycle Sequence Kit (Thermo Fisher Scientific, USA).

Promoter prediction

The promoter regions were predicted by the bacterial sigma-70 promoter recognition program (BPROM) online software (http://www.softberry.com/).

Reverse transcription-quantitative PCR (RT-qPCR)

For the preparation of total RNAs, 270 μL of E. coli cultures after 24 h of culture were mixed with 540 μL of the RNA protect Bacteria Reagent (Qiagen, Germany). After incubation for 5 min, the pellets were recovered by centrifugation for 10 min at 5,000 × g and resuspended in 200 μL of lysis buffer (30 mM Tris-HCl pH 8.0, 1 mM EDTA, and 15 mg/mL lysozyme) containing proteinase K. Seven hundred microliters of buffer RLT (Qiagen, Germany) containing 40 mM DTT were added to the lysed pellets after incubation for 10 min, followed by the purification of total RNAs from the RLT-treated pellets using an RNeasy mini kit (Qiagen, Germany) according to the manufacturer’s protocol. The prepared RNAs were treated with RNase-free DNase I (NEB, USA) for 1 h at 37 °C, and DNase I was heat-denatured for 10 min at 75 °C after the addition of EDTA (final concentration, 5 mM). Next, reverse transcription of DNase-treated RNAs was performed using the High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, USA) for 1 h at 37 °C, followed by denaturation for 5 min at 95 °C. RT-qPCR was performed using the prepared cDNAs and the Power SYBR Green PCR Master Mix on a QuantStudio 3 (Thermo Fisher Scientific, USA). The reaction conditions were as follows: incubation at 50 °C for 2 min and 95 °C for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative expression of the modified TMV cDNAs was calculated via the ΔΔCt method with pUC19-ΔSD-G as a reference using the Design and Analysis software ver. 1.4 software (Thermo Fisher Scientific, USA). The E. coli siroheme synthase (cysG) was used as a housekeeping gene for normalization.

Statistical analysis

All experimental analyses were performed in biological triplicate. The Welch t test was used to determine the significance of the difference between the means of two groups.

Results

Design of modified TMV cDNAs

To prepare the vectors for the modified TMV cDNAs, the regions consisting of the MP gene, the Sgp for CP, the CP-coding genes, and the 3’-UTR of the TMV genome were replaced with cloning sites (ZraI/AscI/AvrII/NotI). Then, the mUkG1-encoding gene was introduced using the ZraI and AscI sites (Fig 1). The DNA fragments used for constructing the vectors are listed in S1 Table. More specifically, the initiation codon of MP in the 3’-end of the RdRp-domain-encoding gene was deleted using synonymous substitution (GATGGC to GACGGC), and a ZraI site was inserted just after the termination codon of the RdRp domain. Four truncated mutants, lacking the region from the 5’-UTR to the N-terminal of the IR, or the N-terminal side of the Hel domain of the TMV replicase, were designed. One of them, pUC-TM5-G, retained the Hel domain but lacked the first half of the IR. The remaining three mutants (pUC-TM7-G, pUC-TM8-G, and pUC-TM9-G) gradually lost the Hel domain (Fig 1). Specifically, pUC-TM7-G and pUC-TM8-G lost the N-terminal region and the first half of the Hel domain, respectively, and pUC-TM9-G included only the C-terminal region of the Hel domain. pUC-G was constructed as the negative control. For all vectors, the modified TMV genes and the mUkG1 gene were introduced into the lac promoter-deleted pUC19 vector in the opposite direction of the ampicillin resistance gene cassette (Amp^R) and replication origin.

Detection of fluorescence intensity in E. coli harboring pUC-TMx-G

As mUkG1 expression can be quantified based on fluorescent intensities, the fluorescence intensities of the E. coli HST08 cells harboring pUC-TMx-Gs were measured at different time points (4, 8, 12, 24, 36, and 48 h) (Fig 2). There was no difference in the fluorescence intensities between pUC-G and the pUC19 mock vector, indicating that mUkG1 was not expressed by pUC-G and an autofluorescence in E. coli. In contrast, pUC-TM-G, pUC-TM5-G, and pUC-TM7-G exhibited significantly higher fluorescence intensities at 8, 12, 24, 36, and 48 h. Notably, the fluorescence intensity of cells harboring pUC-TM-G and pUC-TM5-G was more than 4-fold higher at 24, 36, and 48 h, while that emitted by pUC-TM8-G and pUC-TM9-G was similar to that of pUC-G, although slight differences were observed at 24 and 36 h. The E. coli cell pellets obtained after a 24 h-culture are shown in Fig 3. When these pellets were excited with a blue LED light source, fluorescence emission was observed from pUC-TM-G, pUC-TM5-G, and pUC-TM7-G, whereas that from pUC19, pUC-G, pUC-TM8-G, and pUC-TM9-G was relatively lower. The fluorescence of pUC-TM7-G was lower than that of pUC-TM-G and pUC-TM5-G. These results are consistent with the fluorescence intensity measurements, confirming that mUkG1 was expressed by pUC-TM-G, pUC-TM5-G, and pUC-TM7-G.

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Fig 2. Fluorescence intensity analysis of E. coli harboring the pUC-TMx-G vectors.

Fold changes in fluorescence intensities for each E. coli culture over the control, i.e., pUC-G (white bar with underlined label), were calculated at different cultivation time points (4, 8, 12, 24, 36, and 48 h), as shown. Data are presented as the mean ± standard deviation of three biological replicates. Significant differences from pUC-G are indicated by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

https://doi.org/10.1371/journal.pone.0358128.g002

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Fig 3. Expression analysis of mUkG1 in E. coli harboring the pUC-TMx-G vectors.

Photographs of cell pellets before (up) and after (down) exposure to a fluorescent excitation LED light source after cultivation for 24 h.

https://doi.org/10.1371/journal.pone.0358128.g003

Identification of the initiation codon for mUkG1

Despite the expression of mUkG1 in pUC-TM-G, pUC-TM5-G, and pUC-TM7-G, it was unknown whether mUkG1 proteins were translated from the correct initiation codon. Therefore, the initiation codon (ATG) of mUkG1 was replaced with AGT on pUC-TMx-G, and the fluorescence intensity of these mutants (denoted as pUC-TMx-GΔ) was measured after 24 h cultivation (Fig 4). Unlike pUC-TM-G, pUC-TM5-G and pUC-TM7-G, the fluorescence intensities for pUC-TM-GΔ, pUC-TM5-GΔ and pUC-TM7-GΔ were significantly weak, comparable to those for pUC19, pUC-G, pUC-TM8-G and pUC-TM9-G. Next, a 6×His-tag was fused to the C-terminal of mUkG1 in pUC-TM-G, pUC-TM5-G, pUC-TM7-G, and pUC-TM9-G (denoted as pUC-TMx-GH) for Western blotting analysis using an anti-6×His-tag antibody. After culturing for 24 h, the total protein from the transformants harboring pUC-TM-GH, pUC-TM5-GH, pUC-TM7-GH, and pUC-TM9-GH was analysed by SDS-PAGE and Western blotting. Bands corresponding to mUkG1 could not be identified for any of the transformants using Coomassie brilliant blue staining after SDS-PAGE, probably because of low expression of mUkG1 (Fig 5A). In contrast, Western blotting analysis clearly revealed 25.6-kDa bands corresponding to mUkG1 for pUC-TM-GH, pUC-TM5-GH, and pUC-TM7-GH, but not for pUC-TM9-GH (Fig 5B). These observations suggest that intact mUkG1 proteins with no fused peptide or nontruncation at their N-terminal were expressed; i.e., mUkG1 proteins were translated from the correct initiation codon.

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Fig 4. Comparison of fluorescence intensity exhibited by E. coli harboring pUC-TMx-G and pUC-TMx-GΔ.

Fold changes in fluorescence intensities over the control, i.e., pUC-G (white bar with underlined label), were calculated after 24 h of incubation for each E. coli transformant. Data are presented as the mean ± standard deviation of three biological replicates. Significant differences from pUC-G are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.

https://doi.org/10.1371/journal.pone.0358128.g004

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Fig 5. SDS-PAGE and Western blotting analyses of 6×His-tagged mUkG1 expressed in E. coli harboring pUC-TMx-GH after 24 h of culture.

After measuring OD₆₀₀, the cell pellet was collected from 1.5 mL of the culture in LB medium and lysed in 75 µL of BugBuster Master Mix. A 20 µL sample was prepared from each lysate, normalized to 8.5 divided by the OD₆₀₀ value. Using 10 µl of each sample, (A) SDS-PAGE and (B) Western blotting analyses were performed using 2 µL of the lysate of the BL21 (DE3) E. coli strain expressing 6×His-tagged mUkG1 as the positive control. The 6×His-tagged mUkG1 is indicated by the solid arrowhead.

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Promoter analysis

All the plasmids constructed in this study lost the lacZ promoter derived from pUC19. Furthermore, because the modified TMV genes were cloned in a direction opposite to that of Amp^R, the mUkG1 sense RNA was not transcribed even if the transcription readthrough for Amp^R expression occurs. This observation led to the hypothesis that the gene expression elements are located upstream of the mUkG1 gene in the modified TMV gene sequence, thereby leading to the transcription of mUkG1 mRNA. To confirm this hypothesis, a promoter prediction analysis was performed using the bacterial sigma-70 promoter recognition program (BPROM), yielding 80% accuracy and specificity [19]. Of the 12 putative promoter candidates (Table 1), one was located at the boundary region of TM7 and TM8 in the Hel domain, and therefore, was present only in pUC-TM-G, pUC-TM5-G, and pUC-TM7-G, which clearly expressed mUkG1 (Fig 6). The linear discriminant function (LDF) score was 4.13 (an LDF score ≥ 0.2 is considered significant) and the suggested core promoter elements were ‘TTGATG’ (−35 box) and ‘TGTTAATTT’ (-10 box). Four candidates with LDF scores of 4.37, 3.94, 2.86, and 2.68 were found in all modified TMV genomes, whereas three candidates with LDF scores of 2.69, 1.70, and 3.62 were detected only in pUC-TM-G and pUC-TM5-G. The remaining four candidates were found only in pUC-TM-G. To determine the 5’-end of the transcribed mRNA, 5’-RACE was also performed with mRNAs derived from pUC-TM5-G and pUC-TM8-G (S2 Fig). The 5’-ends of the PCR amplicons generated using the 5’-end-phosphorylated primer against the mUkG1 sequence, which were over 1 kb from the initiation codon of mUkG1, were barely detectable. This might be because the amplicon length exceeds the maximum elongation capacity of the reverse transcriptase. Therefore, 5’-RACE was performed again using a 5’-end-phosphorylated primer designed to anneal at sequences of 1877 nts upstream of the initiation codon for mUkG1. The 5’-ends located in the TM8 region (2810–3203 nts) were newly detected for pUC-TM5-G (S3 Fig). The most frequently detected 5’ end was at the 2828 nt position, which was located immediately downstream of the predicted promoter at the boundary region of TM7 and TM8.

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Table 1. Promoter prediction using the bacterial sigma-70 promoter recognition program.

https://doi.org/10.1371/journal.pone.0358128.t001

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Fig 6. Prediction of sigma 70 promoters using the bacterial sigma-70 promoter recognition program (BPROM).

The bent arrows indicate the transcription start sites of the predicted promoters. The solid and double lines indicate the regions within pUC-TM-G only and pUC-TM-G and pUC-TM5-G, respectively. The thick lines indicate the regions within pUC-TM-G, pUC-TM5-G, and pUC-TM7-G, while the dashed lines show the areas in all constructs. The core elements and LDF score of the candidate promoter are shown in the box.

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Next, the transcriptional activity of the promoter identified in the cDNA was compared with that of the lac promoter from pUC19. Two reference vectors, pUC19-SD-G and pUC19-ΔSD-G, were constructed by replacing the sequence from the initiation codon to the XbaI site of lacZα in pUC19 with the mUkG1-encoding sequence (S4 Fig). In pUC19-ΔSD-G, the SD sequence (AGGAA) was replaced with its complementary sequence (TCCTT). RT-qPCR analysis confirmed promoter activity in pUC-TM-G, pUC-TM5-G, and pUC-TM7-G, and the transcriptional activity was not significantly different from that of the lac promoter (Fig 7). When the fluorescence intensities of E. coli HST08 cells harboring the two reference vectors were compared with those of cells harboring pUC-TMx-G, pUC19-SD-G showed a 24-fold higher fluorescence intensity, whereas pUC19-ΔSD-G showed a 1.6-fold higher fluorescence intensity than pUC-TM-G and pUC-TM5-G (Fig 8). These observations indicate that the reduced translational output is attributable not to promoter activity but rather to the absence of the SD sequence and the extremely long 5’-UTR.

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Fig 7. RT-qPCR analysis of the mRNAs derived from E. coli harboring pUC-TMx-G, pUC-TMx-GΔ, pUC19-SD-G and pUC19-ΔSD-G.

The relative expression levels of mRNAs for mUkG1 over the control, i.e., pUC19-SD-G (green bar with underlined label), were calculated using the ΔΔCt method. Data are presented as the mean ± standard deviation of three biological replicates. Significant differences from pUC-G are indicated by **P < 0.01, ***P < 0.001 and ****P < 0.0001.

https://doi.org/10.1371/journal.pone.0358128.g007

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Fig 8. Comparison of fluorescence intensity exhibited by E. coli harboring pUC-TMx-G, pUC-TMx-GΔ, pUC19-SD-G and pUC19-ΔSD-G.

Fold changes in fluorescence intensities over the control, i.e., pUC-G (white bar with underlined label), were calculated after 24 h of incubation for each E. coli transformant. Data are presented as the mean ± standard deviation of three biological replicates. Significant differences from pUC-G are indicated by ****P < 0.0001.

https://doi.org/10.1371/journal.pone.0358128.g008

Transcription of modified cDNAs of the TMV genome by inducible promoters

To confirm mUkG1 translation from long mRNAs, which are transcribed from 2096 nts upstream of the initiation codon for mUkG1, bacteriophage T7 promoter-inducible vectors were constructed (Fig 9). All the expression vectors, pT7-TM5-G, pT7-TM7-G, and pT7-TM₂₇₉₁-G, contain the predicted promoter sequence in the Hel domain, while the pT7-TM₂₈₂₈-G, pT7-TM8-G, pT7-TM₄₃₄₁-G and pT7-TM₄₇₅₉-G vectors do not. Based on the 5’-RACE results, pT7-TM₂₈₂₈-G, pT7-TM₄₃₄₁-G, and pT7-TM₄₇₅₉-G were constructed to generate mRNAs transcribed from the detected 5’-ends. pT7-SD-G and pT7 were used as positive and negative controls, respectively. Constructs harboring mutated initiation codons included pT7-TM_2828-GΔ, pT7-TM₄₃₄₁-GΔ, and pT7-TM₄₇₅₉-GΔ. These vectors were transformed into the E. coli KRX strain, which was cultivated in LB medium. Fluorescence intensity was measured under both uninduced (after 10 h of culture) and induced (6 h after rhamnose induction following a 4-h culture period) conditions. Under the no-induction condition, significant fluorescence intensity was detected from pT7-TM5-G, pT7-TM7-G, pT7-TM₂₇₉₁-G, and pT7-SD-G. pT7-TM8-G, pT7-TM₂₈₂₈-G, pT7-TM₄₃₄₁-G, pT7-TM₄₇₅₉-G, pT7-TM_2828-GΔ, pT7-TM₄₃₄₁-GΔ, pT7-TM₄₇₅₉-GΔ did not exhibit fluorescence (Fig 10A). Probably, the fluorescens for pT7-SD-G is from basal expression of mUkG1. As expected, pT7-TM₂₇₉₁-G, which contains the predicted promoter, exhibited fluorescence emission despite a difference of only 20 nts between pT7-TM₂₇₉₁-G and pT7-TM8-G. Under the induced condition, fluorescence intensity increased for pT7-TM₂₈₂₈-G and pT7-TM₄₃₄₁-G in addition to pT7-TM5-G, pT7-TM7-G, pT7-TM₂₇₉₁-G, and pT7-SD-G. In contrast, no increase in fluorescence intensity was observed for pT7-TMx-GΔs. These results indicate that the inducible promoter enhances mRNA transcription, resulting in an increased expression of mUkG1 along with the translation of mUkG1 from the correct initiation codon, irrespective of the length of 5’-UTR (Fig 10B). The vectors, pT7-TM8-G and pT7-TM₄₇₅₉-G, did not display fluorescence even after induction. It might be because pT7-TM8-G and TM₄₇₅₉-G have a TT sequence at positions +2 and +3 of the T7 promoter, which showed significantly lower activity in the T7 promoter variants randomized from positions +2 to +16 [20]. In a preliminary examination, pT7-TM₂₈₀₉-G and pT7-TM₄₇₅₈-G, which harbor the GT sequence at positions +2 and +3, showed increased fluorescence intensity upon induction (S5 Fig).

Download:

Fig 9. Construction of the pT7-TMx-G vectors.

The fragments of the 5’-end-truncated mutants and SD-attached mUkG1 were cloned just downstream of the T7 promoter in the pET26b backbone.

https://doi.org/10.1371/journal.pone.0358128.g009

Download:

Fig 10. Fluorescence intensity analysis of E. coli harboring the pT7-TMx-G vectors under uninduced and induced conditions.

Fold changes in fluorescence intensities over the control, pT7 (black bar with underlined label), were calculated for each E. coli transformant after 10 h of culture under (A) uninduced and (B) induced (6 h after rhamnose induction following a 4-h culture period) conditions. Data are presented as the mean ± standard deviation of three biological replicates. Significant differences from the control are indicated by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

https://doi.org/10.1371/journal.pone.0358128.g010

Discussion

In this study, cDNA fragments were generated from the TMV RNA genome by replacing the MP gene and its downstream region with a gene encoding the green fluorescent protein mUkG1. Intact mUkG1 proteins were expressed from pUC-TM-G and pUC-TM7-G without additional or missing amino acids. Moreover, the Hel domain-coding region contributed to mUkG1 expression in E. coli and was predicted to contain a sigma 70 promoter with an LDF score of 4.13. Because the mRNA derived from this promoter contributes to mUkG1 expression, the 5’-UTR is expected to span 2096 nts, which is extremely long. In E. coli, the 5’-UTR is usually 25–35 nts long [21]. Expression analyses using pUC-TMx-G and pT7-TMx-G demonstrated that the predicted promoter functions appropriately and that intact mUkG1 is expressed irrespective of the length of the 5’-UTR of the mUkG1 mRNA.

SD-independent translation in E. coli has been reported in the RNA genomes of several plant RNA viruses, such as TMV RNA and Alfalfa Mosaic Virus RNA 4 [16,17]. The Ω- and U-rich sequences in the 5’-UTRs are recognized by the ribosomal protein S1 of E. coli, resulting in SD-independent translation [17]. S1 has high affinity for U-rich or A/U-rich sequences [22,23] and supports the binding of the 30S subunit to mRNAs without requiring an SD sequence [24]. Bioinformatics analyses have shown that the sequences surrounding the initiation codons of mRNAs without an SD sequence form significantly weaker secondary structures than those with an SD sequence [25,26]. In the modified TMV cDNA, the 80 nts (containing RdRp-encoding gene) upstream of the initiation codon of mUkG1 had an AT content of 70%, which was significantly higher than the E. coli genome, which has approximately 49% AT content [27–29]. In addition, analysis of SD sequence 4-mers (AGGA|GGAG|GAGG), 5-mers (AGGAG|GGAGG), or 6-mers (AGGAGG) indicated that there was only one SD sequence, GGAGG, at 111 nts upstream of the initiation codon. This GGAGG sequence was located in the Sgp region of MP in the RdRp-encoding gene, where the negative strand forms two stem-loop structures [10]. This led to the prediction of a stem-loop structure involving GGAGG in the positive strand. Therefore, the lower expression of intact mUkG1 may have been driven in an SD-independent manner. Additionally, the stem-loop structures and adjacent A/U-rich region may function as a ribosomal standby site [30], as the rhamnose-induced increase in fluorescence intensity was significantly lower for pT7-TM₄₇₅₈-G (S5 Fig), in which 15 nts at the 5’-end of the MP Sgp region were deleted, potentially destabilizing the stem-loop structures.

Some eukaryotic promoters, such as the B33 promoter and the ST-LSI promoter of Solanum tuberosum, can be recognized by E. coli [31,32]. Moreover, random DNA sequences from S. cerevisiae can initiate gene expression in E. coli [33]. Studies have shown that the promoters of eukaryotic viruses, such as Cauliflower mosaic virus, Simian vacuolating virus 40, and human immunodeficiency virus type 1, can drive gene expression in E. coli [32,34]. These viruses are either DNA or RNA viruses with a reverse transcriptase (RT) that requires the transcription of DNA into RNA to replicate in host cells. In turn, TMV is an RNA virus that lacks RT and does not require a DNA intermediate for replication. Therefore, it was surprising that the promoter region recognized by E. coli in the cDNA of the TMV RNA genome is functional. These results indicate that the genomes of RNA viruses potentially hide gene expression elements that function in prokaryotic cells.

The crystal structure of ToMV-Hel, revealed using X-ray crystallography, was shown to consist of an N-terminal domain and two RecA-like domains (denoted as 1A and 2A), which form a helicase core [35]. As the primary amino acid sequences of TMV-Hel and ToMV-Hel demonstrate 89.6% homology, a structural model of TMV-Hel has been constructed based on the ToMY-Hel structure [36]. In this model, the predicted promoter is located just downstream of the putative Walker B motif (RLFIDE at nts 2772–2789) of the RecA-like 1A domain: 261 nts upstream of the encoding gene of the RecA-like 2A domain, immediately followed by the RdRp domain. RecA mediates homologous recombination and genetic repair in almost all bacteria. [37,38]. RecA and RecA-like proteins have been found in several prokaryotes and eukaryotes and are believed to have evolved from a single common ancestor of cellular organisms [39].

Among RNA viruses, the RdRp-encoding genes are the only universally conserved genes. The viral RdRps belong to polymerases containing palm catalytic domains [40,41]. The phylogenetic tree constructed for RdRp based on multiple-sequence alignments suggested that the last common ancestors of +RNA viruses encoded only two proteins, the RdRp and a single jelly-roll CP, and that additional genes (such as helicase- and capping enzyme-encoding genes) were independently captured in subsequent evolution steps [4]. Furthermore, the RdRps of +RNA viruses and the RTs of group II introns, which are widespread in bacterial genomes, were shown to be highly similar [1,4,42]. In the TMV genome, a termination codon was inserted between the MT-Hel and the RdRp domains, implying that the functions of MT and Hel were acquired separately from the RdRp in different evolutionary steps. The extremely long 5’-UTR may arise from a coincidental combination of the promoter trace with an A/T-rich sequence in the 3’-terminal region of the RdRp-coding gene. Taken together, the prokaryotic characteristics in the TMV genome are as follows: (i) the promoter sequence was hidden upstream of Rec-like 2A domain of TMV-Hel, (ii) a local RNA unfolding mechanism, mediated by A/T-rich sequences, potentially contributed to the recognition of the correct initiation codon of mUkG1 (as substitute for MP) without an SD sequence in E. coli, and (iii) the RdRps of RNA viruses might share a common ancestor with RTs in group II introns. These features suggest that ancestral prokaryotic gene elements significantly contribute to the origin and evolution of highly diverse and abundant RNA viruses found in the virome of eukaryotes. Recent metagenomic studies suggest a much greater diversity of RNA viruses in prokaryotes than that described previously [43,44]. In addition, an evolutionary relationship between arthropod and plant viruses belonging to the families Virgaviridae and Kitaviridae has been proposed [45]. Bacterial endosymbionts are widespread in insects, particularly in sap-feeding species, many of which serve as vectors of plant diseases [46–48]. Thus, the presence of an E. coli-recognizable promoter sequence in the TMV genome may reflect evolutionary processes involving horizontal gene transfer among arthropods, their microbial partners, and viruses. Analysis of the genome structure of newly discovered prokaryotic RNA viruses [49,50] and hidden gene elements in RNA genomes using cDNAs would facilitate a deeper understanding of the origin and evolution of RNA viruses.

Supporting information

S1 Fig. Schematic representation of the vector construction strategy.

(A) A fragment of the mUkG1-encoding gene was digested using AscI and then cloned into the ZraI and AscI sites of pCLY-TM. (B) Next, the PCR products of the modified TMV gene, its truncated mutants, and mUkG1 were digested using AvrII and cloned into the SmaI and XbaI sites of pUC19. (C) Finally, the fragments of the modified TMV gene, its truncated mutants, and mUkG1 were amplified by PCR using primers with 15-base extensions complementary to the vector. The PCR fragments were cloned into pUC19 between both PvuII sites using the In-Fusion cloning kit.

https://doi.org/10.1371/journal.pone.0358128.s001

(TIF)

S2 Fig. 5’-RACE analysis for pUC-TM5-G and pUC-TM8-G using a primer specific to the mUkG1 sequence.

The 5’-ends of the cDNA sequences from 73 and 61 clones for pUC-TM5-G and pUC-TM8-G, respectively, were determined. The bent and blue arrows indicate the nucleotide and annealing positions of the 5’-end-phosphorylated primer, respectively.

https://doi.org/10.1371/journal.pone.0358128.s002

(TIF)

S3 Fig. 5’-RACE analysis for pUC-TM5-G and pUC-TM8-G using a primer specific to 1877 nts upstream of the initiation codon of mUkG1.

The 5’-ends of the cDNAs from 43 and 31 clones for pUC-TM5-G and pUC-TM8-G, respectively, were determined. The bent and blue arrows show the nucleotide and annealing positions of the 5’-end-phosphorylated primer, respectively.

https://doi.org/10.1371/journal.pone.0358128.s003

(TIF)

S4 Fig. Construction of the pUC19-SD-G and pUC19-ΔSD-G vectors.

The N-terminal region of the LacZα peptide in the pUC19 was replaced with mUkG1. In pUC19-ΔSD-G, the SD sequence was replaced with its complementary sequence.

https://doi.org/10.1371/journal.pone.0358128.s004

(TIF)

S5 Fig. Fluorescence intensity analysis of E. coli harboring pT7-TM₂₈₀₉-G, TM8-G, TM₄₇₅₈-G, TM₄₇₅₉-G, and SD-G vectors under uninduced and induced conditions.

Fold changes in fluorescence intensities over the control, pT7 (black bar with underlined label), were calculated for each E. coli transformant after 10 h of culture under (A) uninduced and (B) induced (6 h after rhamnose induction following a 4-h culture period) conditions. Data are presented as the mean ± standard deviation of three biological replicates. Significant differences from the control are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.

https://doi.org/10.1371/journal.pone.0358128.s005

(TIF)

S1 Table. Sequences of TMV fragments and mUkG1.

Homologous sequences between two fragments connected by homologous recombination and restriction enzyme sites are indicated by the underlined texts and grey boxes, respectively.

https://doi.org/10.1371/journal.pone.0358128.s006

(DOCX)

S2 Table. Primer list.

The primers used to construct different vectors and for 5’-RACE and RT-qPCR analysis are listed.

https://doi.org/10.1371/journal.pone.0358128.s007

(DOCX)

S3 Table. Promoter sequences.

The T7 promoter was connected immediately upstream of each cDNA without any nucleotide insertions.

https://doi.org/10.1371/journal.pone.0358128.s008

(DOCX)

S1 Raw images. Original gel image and blot for Fig 5.

https://doi.org/10.1371/journal.pone.0358128.s009

(PDF)

S1 Dataset. All plotted datapoints, organized by figure in Excel file.

https://doi.org/10.1371/journal.pone.0358128.s010

(XLSX)

Acknowledgments

The author would like to thank Enago (www.enago.jp) for the English language review.

References

1. Koonin EV, Dolja VV, Krupovic M, Varsani A, Wolf YI, Yutin N, et al. Global Organization and Proposed Megataxonomy of the Virus World. Microbiol Mol Biol Rev. 2020;84(2):e00061-19. pmid:32132243
- View Article
- PubMed/NCBI
- Google Scholar
2. Koonin EV, Dolja VV, Krupovic M. Origins and evolution of viruses of eukaryotes: The ultimate modularity. Virology. 2015;479–480:2–25. pmid:25771806
- View Article
- PubMed/NCBI
- Google Scholar
3. Dolja VV, Koonin EV. Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer. Virus Res. 2018;244:36–52. pmid:29103997
- View Article
- PubMed/NCBI
- Google Scholar
4. Wolf YI, Kazlauskas D, Iranzo J, Lucía-Sanz A, Kuhn JH, Krupovic M, et al. Origins and Evolution of the Global RNA Virome. mBio. 2018;9(6):e02329-18. pmid:30482837
- View Article
- PubMed/NCBI
- Google Scholar
5. Reguera J, Gerlach P, Cusack S. Towards a structural understanding of RNA synthesis by negative strand RNA viral polymerases. Curr Opin Struct Biol. 2016;36:75–84. pmid:26826467
- View Article
- PubMed/NCBI
- Google Scholar
6. Ahlquist P. Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses. Nat Rev Microbiol. 2006;4(5):371–82. pmid:16582931
- View Article
- PubMed/NCBI
- Google Scholar
7. Goelet P, Lomonossoff GP, Butler PJ, Akam ME, Gait MJ, Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982;79(19):5818–22. pmid:6964389
- View Article
- PubMed/NCBI
- Google Scholar
8. Ishikawa M, Okada Y. Replication of tobamovirus RNA. Proc Jpn Acad Ser B. 2004;80(5):215–24. pmid:27296148
- View Article
- PubMed/NCBI
- Google Scholar
9. Kawamura-Nagaya K, Ishibashi K, Huang Y-P, Miyashita S, Ishikawa M. Replication protein of tobacco mosaic virus cotranslationally binds the 5’ untranslated region of genomic RNA to enable viral replication. Proc Natl Acad Sci U S A. 2014;111(16):E1620-8. pmid:24711385
- View Article
- PubMed/NCBI
- Google Scholar
10. Grdzelishvili VZ, Chapman SN, Dawson WO, Lewandowski DJ. Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo. Virology. 2000;275(1):177–92. pmid:11017798
- View Article
- PubMed/NCBI
- Google Scholar
11. Gallie DR. The 5’-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F. Nucleic Acids Res. 2002;30(15):3401–11. pmid:12140325
- View Article
- PubMed/NCBI
- Google Scholar
12. Sleat DE, Gallie DR, Jefferson RA, Bevan MW, Turner PC, Wilson TM. Characterisation of the 5’-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro. Gene. 1987;60(2–3):217–25. pmid:2832252
- View Article
- PubMed/NCBI
- Google Scholar
13. van Belkum A, Abrahams JP, Pleij CW, Bosch L. Five pseudoknots are present at the 204 nucleotides long 3’ noncoding region of tobacco mosaic virus RNA. Nucleic Acids Res. 1985;13(21):7673–86. pmid:3934645
- View Article
- PubMed/NCBI
- Google Scholar
14. Chujo T, Ishibashi K, Miyashita S, Ishikawa M. Functions of the 5’- and 3’-untranslated regions of tobamovirus RNA. Virus Res. 2015;206:82–9. pmid:25683511
- View Article
- PubMed/NCBI
- Google Scholar
15. Coutts RH, Cocking EC, Kassanis B. Infection of protoplasts from yeast with tobacco mosaic virus. Nature. 1972;240(5382):466–7. pmid:4565938
- View Article
- PubMed/NCBI
- Google Scholar
16. Castel A, Kraal B, Konieczny A, Bosch L. Translation by Escherichia coli ribosomes of alfalfa mosaic virus RNA 4 can be initiated at two sites on the monocistronic message. Eur J Biochem. 1979;101(1):123–33. pmid:389629
- View Article
- PubMed/NCBI
- Google Scholar
17. Tzareva NV, Makhno VI, Boni IV. Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions. FEBS Lett. 1994;337(2):189–94. pmid:8287975
- View Article
- PubMed/NCBI
- Google Scholar
18. Roberts MW, Rabinowitz JC. The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes. J Biol Chem. 1989;264(4):2228–35. pmid:2644257
- View Article
- PubMed/NCBI
- Google Scholar
19. Solovyev V, Salamov A. Automatic annotation of microbial genomes and metagenomic sequences. In: Li RW, editor. Metagenomics and its applications in agriculture, biomedicine and environmental studies. Hauppauge, N.Y.: Nova Science Publisher’s. 2011.
20. Conrad T, Plumbom I, Alcobendas M, Vidal R, Sauer S. Maximizing transcription of nucleic acids with efficient T7 promoters. Commun Biol. 2020;3(1):439. pmid:32796901
- View Article
- PubMed/NCBI
- Google Scholar
21. Kim D, Hong JS-J, Qiu Y, Nagarajan H, Seo J-H, Cho B-K, et al. Comparative analysis of regulatory elements between Escherichia coli and Klebsiella pneumoniae by genome-wide transcription start site profiling. PLoS Genet. 2012;8(8):e1002867. pmid:22912590
- View Article
- PubMed/NCBI
- Google Scholar
22. Komarova AV, Tchufistova LS, Supina EV, Boni IV. Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation. RNA. 2002;8(9):1137–47. pmid:12358433
- View Article
- PubMed/NCBI
- Google Scholar
23. Wen J-D, Kuo S-T, Chou H-HD. The diversity of Shine-Dalgarno sequences sheds light on the evolution of translation initiation. RNA Biol. 2021;18(11):1489–500. pmid:33349119
- View Article
- PubMed/NCBI
- Google Scholar
24. Boni IV, Isaeva DM, Musychenko ML, Tzareva NV. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1. Nucleic Acids Res. 1991;19(1):155–62. pmid:2011495
- View Article
- PubMed/NCBI
- Google Scholar
25. Nakagawa S, Niimura Y, Gojobori T. Comparative genomic analysis of translation initiation mechanisms for genes lacking the Shine-Dalgarno sequence in prokaryotes. Nucleic Acids Res. 2017;45(7):3922–31. pmid:28334743
- View Article
- PubMed/NCBI
- Google Scholar
26. Scharff LB, Childs L, Walther D, Bock R. Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites. PLoS Genet. 2011;7(6):e1002155. pmid:21731509
- View Article
- PubMed/NCBI
- Google Scholar
27. Ely B. Genomic GC content drifts downward in most bacterial genomes. PLoS One. 2021;16(5):e0244163. pmid:34038432
- View Article
- PubMed/NCBI
- Google Scholar
28. Matsumura Y, Yamamoto M, Nakano S, Nagao M. Complete Genome Sequence of Escherichia coli ME8067, an Azide-Resistant Laboratory Strain Used for Conjugation Experiments. Genome Announc. 2018;6(25):e00515-18. pmid:29930039
- View Article
- PubMed/NCBI
- Google Scholar
29. Li X-Q, Du D. Variation, evolution, and correlation analysis of C+G content and genome or chromosome size in different kingdoms and phyla. PLoS One. 2014;9(2):e88339. pmid:24551092
- View Article
- PubMed/NCBI
- Google Scholar
30. Romilly C, Deindl S, Wagner EGH. The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5’ structure element. Proc Natl Acad Sci U S A. 2019;116(32):15901–6. pmid:31320593
- View Article
- PubMed/NCBI
- Google Scholar
31. Lewin A, Jacob D, Freytag B, Appel B. Gene Expression in Bacteria Directed by Plant-specific Regulatory Sequences. Transgenic Res. 1998;7(6):403–11.
- View Article
- Google Scholar
32. Jacob D, Lewin A, Meister B, Appel B. Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery. Transgenic Res. 2002;11(3):291–303. pmid:12113461
- View Article
- PubMed/NCBI
- Google Scholar
33. Lewin A, Tran TT, Jacob D, Mayer M, Freytag B, Appel B. Yeast DNA sequences initiating gene expression in Escherichia coli. Microbiol Res. 2004;159(1):19–28. pmid:15160603
- View Article
- PubMed/NCBI
- Google Scholar
34. Lewin A, Mayer M, Chusainow J, Jacob D, Appel B. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli. BMC Biotechnol. 2005;5:19. pmid:15967027
- View Article
- PubMed/NCBI
- Google Scholar
35. Nishikiori M, Sugiyama S, Xiang H, Niiyama M, Ishibashi K, Inoue T, et al. Crystal structure of the superfamily 1 helicase from Tomato mosaic virus. J Virol. 2012;86(14):7565–76. pmid:22573863
- View Article
- PubMed/NCBI
- Google Scholar
36. Li X, Chen K, Gao D, Wang D, Huang M, Zhu H, et al. Binding studies between cytosinpeptidemycin and the superfamily 1 helicase protein of tobacco mosaic virus. RSC Adv. 2018;8(34):18952–8. pmid:35539684
- View Article
- PubMed/NCBI
- Google Scholar
37. Roca AI, Cox MM. RecA protein: structure, function, and role in recombinational DNA repair. Prog Nucleic Acid Res Mol Biol. 1997;56:129–223. pmid:9187054
- View Article
- PubMed/NCBI
- Google Scholar
38. Bell JC, Kowalczykowski SC. RecA: Regulation and Mechanism of a Molecular Search Engine. Trends Biochem Sci. 2016;41(6):491–507. pmid:27156117
- View Article
- PubMed/NCBI
- Google Scholar
39. Lin Z, Kong H, Nei M, Ma H. Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer. Proc Natl Acad Sci U S A. 2006;103(27):10328–33. pmid:16798872
- View Article
- PubMed/NCBI
- Google Scholar
40. Ng KKS, Arnold JJ, Cameron CE. Structure-function relationships among RNA-dependent RNA polymerases. Curr Top Microbiol Immunol. 2008;320:137–56. pmid:18268843
- View Article
- PubMed/NCBI
- Google Scholar
41. te Velthuis AJW. Common and unique features of viral RNA-dependent polymerases. Cell Mol Life Sci. 2014;71(22):4403–20. pmid:25080879
- View Article
- PubMed/NCBI
- Google Scholar
42. Krupovic M, Dolja VV, Koonin EV. Origin of viruses: primordial replicators recruiting capsids from hosts. Nat Rev Microbiol. 2019;17(7):449–58. pmid:31142823
- View Article
- PubMed/NCBI
- Google Scholar
43. Krishnamurthy SR, Janowski AB, Zhao G, Barouch D, Wang D. Hyperexpansion of RNA Bacteriophage Diversity. PLoS Biol. 2016;14(3):e1002409. pmid:27010970
- View Article
- PubMed/NCBI
- Google Scholar
44. Shi M, Lin X-D, Tian J-H, Chen L-J, Chen X, Li C-X, et al. Redefining the invertebrate RNA virosphere. Nature. 2016;540(7634):539–43. pmid:27880757
- View Article
- PubMed/NCBI
- Google Scholar
45. Lu G, Ye Z-X, Qi Y-H, Lu J-B, Mao Q-Z, Zhuo J-C, et al. Endogenous nege-like viral elements in arthropod genomes reveal virus-host coevolution and ancient history of two plant virus families. J Virol. 2024;98(10):e0099724. pmid:39212930
- View Article
- PubMed/NCBI
- Google Scholar
46. Moran NA, Tran P, Gerardo NM. Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes. Appl Environ Microbiol. 2005;71(12):8802–10. pmid:16332876
- View Article
- PubMed/NCBI
- Google Scholar
47. Alarcón ME, Polo PG, Akyüz SN, Rafiqi AM. Evolution and ontogeny of bacteriocytes in insects. Front Physiol. 2022;13:1034066. pmid:36505058
- View Article
- PubMed/NCBI
- Google Scholar
48. Wu W, Shan H-W, Li J-M, Zhang C-X, Chen J-P, Mao Q. Roles of Bacterial Symbionts in Transmission of Plant Virus by Hemipteran Vectors. Front Microbiol. 2022;13:805352. pmid:35154053
- View Article
- PubMed/NCBI
- Google Scholar
49. Krishnamurthy SR, Wang D. Extensive conservation of prokaryotic ribosomal binding sites in known and novel picobirnaviruses. Virology. 2018;516:108–14. pmid:29346073
- View Article
- PubMed/NCBI
- Google Scholar
50. Boros Á, Polgár B, Pankovics P, Fenyvesi H, Engelmann P, Phan TG, et al. Multiple divergent picobirnaviruses with functional prokaryotic Shine-Dalgarno ribosome binding sites present in cloacal sample of a diarrheic chicken. Virology. 2018;525:62–72. pmid:30245195
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Koonin EV, Dolja VV, Krupovic M, Varsani A, Wolf YI, Yutin N, et al. Global Organization and Proposed Megataxonomy of the Virus World. Microbiol Mol Biol Rev. 2020;84(2):e00061-19. pmid:32132243
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Koonin EV, Dolja VV, Krupovic M. Origins and evolution of viruses of eukaryotes: The ultimate modularity. Virology. 2015;479–480:2–25. pmid:25771806
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Dolja VV, Koonin EV. Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer. Virus Res. 2018;244:36–52. pmid:29103997
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Wolf YI, Kazlauskas D, Iranzo J, Lucía-Sanz A, Kuhn JH, Krupovic M, et al. Origins and Evolution of the Global RNA Virome. mBio. 2018;9(6):e02329-18. pmid:30482837
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Reguera J, Gerlach P, Cusack S. Towards a structural understanding of RNA synthesis by negative strand RNA viral polymerases. Curr Opin Struct Biol. 2016;36:75–84. pmid:26826467
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Ahlquist P. Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses. Nat Rev Microbiol. 2006;4(5):371–82. pmid:16582931
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Goelet P, Lomonossoff GP, Butler PJ, Akam ME, Gait MJ, Karn J. Nucleotide sequence of tobacco mosaic virus RNA. Proc Natl Acad Sci U S A. 1982;79(19):5818–22. pmid:6964389
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Ishikawa M, Okada Y. Replication of tobamovirus RNA. Proc Jpn Acad Ser B. 2004;80(5):215–24. pmid:27296148
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Kawamura-Nagaya K, Ishibashi K, Huang Y-P, Miyashita S, Ishikawa M. Replication protein of tobacco mosaic virus cotranslationally binds the 5’ untranslated region of genomic RNA to enable viral replication. Proc Natl Acad Sci U S A. 2014;111(16):E1620-8. pmid:24711385
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Grdzelishvili VZ, Chapman SN, Dawson WO, Lewandowski DJ. Mapping of the Tobacco mosaic virus movement protein and coat protein subgenomic RNA promoters in vivo. Virology. 2000;275(1):177–92. pmid:11017798
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Gallie DR. The 5’-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F. Nucleic Acids Res. 2002;30(15):3401–11. pmid:12140325
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Sleat DE, Gallie DR, Jefferson RA, Bevan MW, Turner PC, Wilson TM. Characterisation of the 5’-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro. Gene. 1987;60(2–3):217–25. pmid:2832252
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. van Belkum A, Abrahams JP, Pleij CW, Bosch L. Five pseudoknots are present at the 204 nucleotides long 3’ noncoding region of tobacco mosaic virus RNA. Nucleic Acids Res. 1985;13(21):7673–86. pmid:3934645
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Chujo T, Ishibashi K, Miyashita S, Ishikawa M. Functions of the 5’- and 3’-untranslated regions of tobamovirus RNA. Virus Res. 2015;206:82–9. pmid:25683511
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Coutts RH, Cocking EC, Kassanis B. Infection of protoplasts from yeast with tobacco mosaic virus. Nature. 1972;240(5382):466–7. pmid:4565938
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Castel A, Kraal B, Konieczny A, Bosch L. Translation by Escherichia coli ribosomes of alfalfa mosaic virus RNA 4 can be initiated at two sites on the monocistronic message. Eur J Biochem. 1979;101(1):123–33. pmid:389629
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Tzareva NV, Makhno VI, Boni IV. Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions. FEBS Lett. 1994;337(2):189–94. pmid:8287975
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Roberts MW, Rabinowitz JC. The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes. J Biol Chem. 1989;264(4):2228–35. pmid:2644257
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Solovyev V, Salamov A. Automatic annotation of microbial genomes and metagenomic sequences. In: Li RW, editor. Metagenomics and its applications in agriculture, biomedicine and environmental studies. Hauppauge, N.Y.: Nova Science Publisher’s. 2011.

[ref20] 20. Conrad T, Plumbom I, Alcobendas M, Vidal R, Sauer S. Maximizing transcription of nucleic acids with efficient T7 promoters. Commun Biol. 2020;3(1):439. pmid:32796901
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref21] 21. Kim D, Hong JS-J, Qiu Y, Nagarajan H, Seo J-H, Cho B-K, et al. Comparative analysis of regulatory elements between Escherichia coli and Klebsiella pneumoniae by genome-wide transcription start site profiling. PLoS Genet. 2012;8(8):e1002867. pmid:22912590
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref22] 22. Komarova AV, Tchufistova LS, Supina EV, Boni IV. Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation. RNA. 2002;8(9):1137–47. pmid:12358433
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref23] 23. Wen J-D, Kuo S-T, Chou H-HD. The diversity of Shine-Dalgarno sequences sheds light on the evolution of translation initiation. RNA Biol. 2021;18(11):1489–500. pmid:33349119
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref24] 24. Boni IV, Isaeva DM, Musychenko ML, Tzareva NV. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1. Nucleic Acids Res. 1991;19(1):155–62. pmid:2011495
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref25] 25. Nakagawa S, Niimura Y, Gojobori T. Comparative genomic analysis of translation initiation mechanisms for genes lacking the Shine-Dalgarno sequence in prokaryotes. Nucleic Acids Res. 2017;45(7):3922–31. pmid:28334743
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref26] 26. Scharff LB, Childs L, Walther D, Bock R. Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites. PLoS Genet. 2011;7(6):e1002155. pmid:21731509
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref27] 27. Ely B. Genomic GC content drifts downward in most bacterial genomes. PLoS One. 2021;16(5):e0244163. pmid:34038432
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref28] 28. Matsumura Y, Yamamoto M, Nakano S, Nagao M. Complete Genome Sequence of Escherichia coli ME8067, an Azide-Resistant Laboratory Strain Used for Conjugation Experiments. Genome Announc. 2018;6(25):e00515-18. pmid:29930039
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref29] 29. Li X-Q, Du D. Variation, evolution, and correlation analysis of C+G content and genome or chromosome size in different kingdoms and phyla. PLoS One. 2014;9(2):e88339. pmid:24551092
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref30] 30. Romilly C, Deindl S, Wagner EGH. The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5’ structure element. Proc Natl Acad Sci U S A. 2019;116(32):15901–6. pmid:31320593
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref31] 31. Lewin A, Jacob D, Freytag B, Appel B. Gene Expression in Bacteria Directed by Plant-specific Regulatory Sequences. Transgenic Res. 1998;7(6):403–11.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref32] 32. Jacob D, Lewin A, Meister B, Appel B. Plant-specific promoter sequences carry elements that are recognised by the eubacterial transcription machinery. Transgenic Res. 2002;11(3):291–303. pmid:12113461
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref33] 33. Lewin A, Tran TT, Jacob D, Mayer M, Freytag B, Appel B. Yeast DNA sequences initiating gene expression in Escherichia coli. Microbiol Res. 2004;159(1):19–28. pmid:15160603
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref34] 34. Lewin A, Mayer M, Chusainow J, Jacob D, Appel B. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli. BMC Biotechnol. 2005;5:19. pmid:15967027
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref35] 35. Nishikiori M, Sugiyama S, Xiang H, Niiyama M, Ishibashi K, Inoue T, et al. Crystal structure of the superfamily 1 helicase from Tomato mosaic virus. J Virol. 2012;86(14):7565–76. pmid:22573863
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref36] 36. Li X, Chen K, Gao D, Wang D, Huang M, Zhu H, et al. Binding studies between cytosinpeptidemycin and the superfamily 1 helicase protein of tobacco mosaic virus. RSC Adv. 2018;8(34):18952–8. pmid:35539684
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref37] 37. Roca AI, Cox MM. RecA protein: structure, function, and role in recombinational DNA repair. Prog Nucleic Acid Res Mol Biol. 1997;56:129–223. pmid:9187054
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref38] 38. Bell JC, Kowalczykowski SC. RecA: Regulation and Mechanism of a Molecular Search Engine. Trends Biochem Sci. 2016;41(6):491–507. pmid:27156117
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref39] 39. Lin Z, Kong H, Nei M, Ma H. Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer. Proc Natl Acad Sci U S A. 2006;103(27):10328–33. pmid:16798872
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref40] 40. Ng KKS, Arnold JJ, Cameron CE. Structure-function relationships among RNA-dependent RNA polymerases. Curr Top Microbiol Immunol. 2008;320:137–56. pmid:18268843
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref41] 41. te Velthuis AJW. Common and unique features of viral RNA-dependent polymerases. Cell Mol Life Sci. 2014;71(22):4403–20. pmid:25080879
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref42] 42. Krupovic M, Dolja VV, Koonin EV. Origin of viruses: primordial replicators recruiting capsids from hosts. Nat Rev Microbiol. 2019;17(7):449–58. pmid:31142823
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref43] 43. Krishnamurthy SR, Janowski AB, Zhao G, Barouch D, Wang D. Hyperexpansion of RNA Bacteriophage Diversity. PLoS Biol. 2016;14(3):e1002409. pmid:27010970
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref44] 44. Shi M, Lin X-D, Tian J-H, Chen L-J, Chen X, Li C-X, et al. Redefining the invertebrate RNA virosphere. Nature. 2016;540(7634):539–43. pmid:27880757
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref45] 45. Lu G, Ye Z-X, Qi Y-H, Lu J-B, Mao Q-Z, Zhuo J-C, et al. Endogenous nege-like viral elements in arthropod genomes reveal virus-host coevolution and ancient history of two plant virus families. J Virol. 2024;98(10):e0099724. pmid:39212930
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

[ref46] 46. Moran NA, Tran P, Gerardo NM. Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes. Appl Environ Microbiol. 2005;71(12):8802–10. pmid:16332876
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

[ref47] 47. Alarcón ME, Polo PG, Akyüz SN, Rafiqi AM. Evolution and ontogeny of bacteriocytes in insects. Front Physiol. 2022;13:1034066. pmid:36505058
View Article
PubMed/NCBI
Google Scholar

[182] View Article

[183] PubMed/NCBI

[184] Google Scholar

[ref48] 48. Wu W, Shan H-W, Li J-M, Zhang C-X, Chen J-P, Mao Q. Roles of Bacterial Symbionts in Transmission of Plant Virus by Hemipteran Vectors. Front Microbiol. 2022;13:805352. pmid:35154053
View Article
PubMed/NCBI
Google Scholar

[186] View Article

[187] PubMed/NCBI

[188] Google Scholar

[ref49] 49. Krishnamurthy SR, Wang D. Extensive conservation of prokaryotic ribosomal binding sites in known and novel picobirnaviruses. Virology. 2018;516:108–14. pmid:29346073
View Article
PubMed/NCBI
Google Scholar

[190] View Article

[191] PubMed/NCBI

[192] Google Scholar

[ref50] 50. Boros Á, Polgár B, Pankovics P, Fenyvesi H, Engelmann P, Phan TG, et al. Multiple divergent picobirnaviruses with functional prokaryotic Shine-Dalgarno ribosome binding sites present in cloacal sample of a diarrheic chicken. Virology. 2018;525:62–72. pmid:30245195
View Article
PubMed/NCBI
Google Scholar

[194] View Article

[195] PubMed/NCBI

[196] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Recombinant strains and growth conditions used for vector construction and mUkG1 expression

Vector construction

Expression analysis of mUkG1

Rapid amplification of cDNA 5’-end (5’-RACE)

Promoter prediction

Reverse transcription-quantitative PCR (RT-qPCR)

Statistical analysis

Results

Design of modified TMV cDNAs

Detection of fluorescence intensity in E. coli harboring pUC-TMx-G

Identification of the initiation codon for mUkG1

Promoter analysis

Transcription of modified cDNAs of the TMV genome by inducible promoters

Discussion

Supporting information

S1 Fig. Schematic representation of the vector construction strategy.

S2 Fig. 5’-RACE analysis for pUC-TM5-G and pUC-TM8-G using a primer specific to the mUkG1 sequence.

S3 Fig. 5’-RACE analysis for pUC-TM5-G and pUC-TM8-G using a primer specific to 1877 nts upstream of the initiation codon of mUkG1.

S4 Fig. Construction of the pUC19-SD-G and pUC19-ΔSD-G vectors.

S5 Fig. Fluorescence intensity analysis of E. coli harboring pT7-TM2809-G, TM8-G, TM4758-G, TM4759-G, and SD-G vectors under uninduced and induced conditions.

S1 Table. Sequences of TMV fragments and mUkG1.

S2 Table. Primer list.

S3 Table. Promoter sequences.

S1 Raw images. Original gel image and blot for Fig 5.

S1 Dataset. All plotted datapoints, organized by figure in Excel file.

Acknowledgments

References

S5 Fig. Fluorescence intensity analysis of E. coli harboring pT7-TM₂₈₀₉-G, TM8-G, TM₄₇₅₈-G, TM₄₇₅₉-G, and SD-G vectors under uninduced and induced conditions.