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Functional characterization of the principal sigma factor RpoD of phytoplasmas via an in vitro transcription assay.

Miura C, Komatsu K, Maejima K, Nijo T, Kitazawa Y, Tomomitsu T, Yusa A, Himeno M, Oshima K, Namba S - Sci Rep (2015)

Bottom Line: In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools.The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns.These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

ABSTRACT
Phytoplasmas (class, Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoDOY) could be a key regulator of host switching, because the expression level of rpoDOY was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoDOY-dependent genes and the consensus promoter elements. The assay revealed that RpoDOY regulated some housekeeping, virulence, and host-phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved -35 and -10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the -35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

No MeSH data available.


Related in: MedlinePlus

Effects of nucleotide substitutions in the putative rrnB promoter elements.(a) Schematic representation of mutant templates with substitutions in the putative P2 promoter elements. The open box represents a 400-bp DNA fragment covering the region from 400 to 1 nt upstream of rrnB, which was used as a control template (PrrnB_P2). A series of 15 mutant templates were prepared, with substitutions to GG or AA introduced around the putative P2 –35 promoter element (mt1, mt2, mt3, mt4, and mt5; mt6, mt7, mt8, mt9, and mt10) or around the putative P2 –10 promoter element (mt11, mt12, mt13, mt14, and mt15). ‘+1’ represents the P2 TSS. Positions of putative P2 –35 and –10 promoter elements are boxed. Dots indicate the same sequence as PrrnB_P2. In vitro transcription with templates containing substitutions to GG (b) or AA (c) in the putative P2 –35 promoter element, and substitutions to GG in the putative P2 –10 promoter element (d). RNAPEc–RpoDOY holoenzyme and DNA templates were incubated with NTP including [γ-32P]CTP. White arrowheads indicate the positions of transcripts from each template. Numbers at the bottom of each in vitro transcription lane represent the relative quantification of autoradiography signals measured by using ImageJ software (version 1.47, National Institutes of Health).
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f2: Effects of nucleotide substitutions in the putative rrnB promoter elements.(a) Schematic representation of mutant templates with substitutions in the putative P2 promoter elements. The open box represents a 400-bp DNA fragment covering the region from 400 to 1 nt upstream of rrnB, which was used as a control template (PrrnB_P2). A series of 15 mutant templates were prepared, with substitutions to GG or AA introduced around the putative P2 –35 promoter element (mt1, mt2, mt3, mt4, and mt5; mt6, mt7, mt8, mt9, and mt10) or around the putative P2 –10 promoter element (mt11, mt12, mt13, mt14, and mt15). ‘+1’ represents the P2 TSS. Positions of putative P2 –35 and –10 promoter elements are boxed. Dots indicate the same sequence as PrrnB_P2. In vitro transcription with templates containing substitutions to GG (b) or AA (c) in the putative P2 –35 promoter element, and substitutions to GG in the putative P2 –10 promoter element (d). RNAPEc–RpoDOY holoenzyme and DNA templates were incubated with NTP including [γ-32P]CTP. White arrowheads indicate the positions of transcripts from each template. Numbers at the bottom of each in vitro transcription lane represent the relative quantification of autoradiography signals measured by using ImageJ software (version 1.47, National Institutes of Health).

Mentions: To define the core promoter elements of rrnB, we focused on the putative P2 promoter and performed in vitro transcription assays. We introduced a series of double-base substitutions to GG into the putative P2 –35 and –10 promoter elements of a DNA fragment containing –400 to –1 of rrnB, named PrrnB_P2 (Fig. 2a). In the putative –35 promoter element of P2, the double-base substitution of TT to GG at positions –35 and –34 (mt2) resulted in a drastic reduction of the transcript level to 16% compared with the intact PrrnB_P2 (Fig. 2b), suggesting decreases in activity of the putative P2 promoter due to these mutations. In contrast, nucleotide substitutions at positions –37 and –36 (mt1), or –33 to –28 (mt3, mt4, and mt5), did not alter promoter activity. Similar results were obtained with the substitutions of TT to AA at the same positions (Fig. 2c). These results suggest that TT at positions –35 and –34 is crucial for promoter activity mediating transcription from rrnB P2. In the P2 –10 promoter element, the substitutions at positions from –12 to –7 (mt12, mt13, and mt14) also resulted in a reduction in promoter activity to 6–22% compared with the intact PrrnB_P2 (Fig. 2d). Substitutions at positions –14 and –13 (mt11), and –6 and –5 (mt15), of P2 slightly decreased promoter activity (Fig. 2d). These results suggest that the conserved hexamer, 5′-TATAAT-3′, is crucial for promoter activity. These findings indicate that at a minimum, the upstream sequence 5′-TT-21bp-TATAAT-3′ of rrnB P2 is essential for recognition by RpoDOY. This is supported by the fact that the same sequence exists in the putative P3 promoter elements (–35 5′-TTGCCA-3′ and –10 5′-TATAAT-3′), but not in the putative P1 promoter elements (–35 5′-TTCACA-3′ and –10 5′-TAATCT-3′).


Functional characterization of the principal sigma factor RpoD of phytoplasmas via an in vitro transcription assay.

Miura C, Komatsu K, Maejima K, Nijo T, Kitazawa Y, Tomomitsu T, Yusa A, Himeno M, Oshima K, Namba S - Sci Rep (2015)

Effects of nucleotide substitutions in the putative rrnB promoter elements.(a) Schematic representation of mutant templates with substitutions in the putative P2 promoter elements. The open box represents a 400-bp DNA fragment covering the region from 400 to 1 nt upstream of rrnB, which was used as a control template (PrrnB_P2). A series of 15 mutant templates were prepared, with substitutions to GG or AA introduced around the putative P2 –35 promoter element (mt1, mt2, mt3, mt4, and mt5; mt6, mt7, mt8, mt9, and mt10) or around the putative P2 –10 promoter element (mt11, mt12, mt13, mt14, and mt15). ‘+1’ represents the P2 TSS. Positions of putative P2 –35 and –10 promoter elements are boxed. Dots indicate the same sequence as PrrnB_P2. In vitro transcription with templates containing substitutions to GG (b) or AA (c) in the putative P2 –35 promoter element, and substitutions to GG in the putative P2 –10 promoter element (d). RNAPEc–RpoDOY holoenzyme and DNA templates were incubated with NTP including [γ-32P]CTP. White arrowheads indicate the positions of transcripts from each template. Numbers at the bottom of each in vitro transcription lane represent the relative quantification of autoradiography signals measured by using ImageJ software (version 1.47, National Institutes of Health).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493692&req=5

f2: Effects of nucleotide substitutions in the putative rrnB promoter elements.(a) Schematic representation of mutant templates with substitutions in the putative P2 promoter elements. The open box represents a 400-bp DNA fragment covering the region from 400 to 1 nt upstream of rrnB, which was used as a control template (PrrnB_P2). A series of 15 mutant templates were prepared, with substitutions to GG or AA introduced around the putative P2 –35 promoter element (mt1, mt2, mt3, mt4, and mt5; mt6, mt7, mt8, mt9, and mt10) or around the putative P2 –10 promoter element (mt11, mt12, mt13, mt14, and mt15). ‘+1’ represents the P2 TSS. Positions of putative P2 –35 and –10 promoter elements are boxed. Dots indicate the same sequence as PrrnB_P2. In vitro transcription with templates containing substitutions to GG (b) or AA (c) in the putative P2 –35 promoter element, and substitutions to GG in the putative P2 –10 promoter element (d). RNAPEc–RpoDOY holoenzyme and DNA templates were incubated with NTP including [γ-32P]CTP. White arrowheads indicate the positions of transcripts from each template. Numbers at the bottom of each in vitro transcription lane represent the relative quantification of autoradiography signals measured by using ImageJ software (version 1.47, National Institutes of Health).
Mentions: To define the core promoter elements of rrnB, we focused on the putative P2 promoter and performed in vitro transcription assays. We introduced a series of double-base substitutions to GG into the putative P2 –35 and –10 promoter elements of a DNA fragment containing –400 to –1 of rrnB, named PrrnB_P2 (Fig. 2a). In the putative –35 promoter element of P2, the double-base substitution of TT to GG at positions –35 and –34 (mt2) resulted in a drastic reduction of the transcript level to 16% compared with the intact PrrnB_P2 (Fig. 2b), suggesting decreases in activity of the putative P2 promoter due to these mutations. In contrast, nucleotide substitutions at positions –37 and –36 (mt1), or –33 to –28 (mt3, mt4, and mt5), did not alter promoter activity. Similar results were obtained with the substitutions of TT to AA at the same positions (Fig. 2c). These results suggest that TT at positions –35 and –34 is crucial for promoter activity mediating transcription from rrnB P2. In the P2 –10 promoter element, the substitutions at positions from –12 to –7 (mt12, mt13, and mt14) also resulted in a reduction in promoter activity to 6–22% compared with the intact PrrnB_P2 (Fig. 2d). Substitutions at positions –14 and –13 (mt11), and –6 and –5 (mt15), of P2 slightly decreased promoter activity (Fig. 2d). These results suggest that the conserved hexamer, 5′-TATAAT-3′, is crucial for promoter activity. These findings indicate that at a minimum, the upstream sequence 5′-TT-21bp-TATAAT-3′ of rrnB P2 is essential for recognition by RpoDOY. This is supported by the fact that the same sequence exists in the putative P3 promoter elements (–35 5′-TTGCCA-3′ and –10 5′-TATAAT-3′), but not in the putative P1 promoter elements (–35 5′-TTCACA-3′ and –10 5′-TAATCT-3′).

Bottom Line: In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools.The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns.These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.

ABSTRACT
Phytoplasmas (class, Mollicutes) are insect-transmissible and plant-pathogenic bacteria that multiply intracellularly in both plants and insects through host switching. Our previous study revealed that phytoplasmal sigma factor rpoD of OY-M strain (rpoDOY) could be a key regulator of host switching, because the expression level of rpoDOY was higher in insect hosts than in plant hosts. In this study, we developed an in vitro transcription assay system to identify RpoDOY-dependent genes and the consensus promoter elements. The assay revealed that RpoDOY regulated some housekeeping, virulence, and host-phytoplasma interaction genes of OY-M strain. The upstream region of the transcription start sites of these genes contained conserved -35 and -10 promoter sequences, which were similar to the typical bacterial RpoD-dependent promoter elements, while the -35 promoter elements were variable. In addition, we searched putative RpoD-dependent genes based on these promoter elements on the whole genome sequence of phytoplasmas using in silico tools. The phytoplasmal RpoD seems to mediate the transcription of not only many housekeeping genes as the principal sigma factor, but also the virulence- and host-phytoplasma interaction-related genes exhibiting host-specific expression patterns. These results indicate that more complex mechanisms exist than previously thought regarding gene regulation enabling phytoplasmas to switch hosts.

No MeSH data available.


Related in: MedlinePlus