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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

In vitro transcription with templates containing the promoter region of various gene categories.RNAPEc-RpoDOY and DNA templates were incubated with NTP, including [γ-32P]CTP in the absence (–) or presence (+) of RpoDOY. The length of each template is as follows: PibpA, 713 bp (–512 to +201 of ibpA); PinfC, 702 bp (–500 to +202 of infC); PrplM, 700 bp (–500 to +200 of rplM); PrpsD, 650 bp (–450 to +200 of rpsD); PrpoD, 624 bp (–400 to +224 of rpoD); P157, 500 bp (–300 to +200 of PAM157); P289, 500 bp (–500 to –1 of PAM289); P486, 566 bp (–300 to +266 of PAM486); Ptengu, 420 bp (–300 to +120 of tengu); Pamp, 400 bp (–200 to +200 of amp). White arrowheads indicate the positions of transcripts from each template.
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f3: In vitro transcription with templates containing the promoter region of various gene categories.RNAPEc-RpoDOY and DNA templates were incubated with NTP, including [γ-32P]CTP in the absence (–) or presence (+) of RpoDOY. The length of each template is as follows: PibpA, 713 bp (–512 to +201 of ibpA); PinfC, 702 bp (–500 to +202 of infC); PrplM, 700 bp (–500 to +200 of rplM); PrpsD, 650 bp (–450 to +200 of rpsD); PrpoD, 624 bp (–400 to +224 of rpoD); P157, 500 bp (–300 to +200 of PAM157); P289, 500 bp (–500 to –1 of PAM289); P486, 566 bp (–300 to +266 of PAM486); Ptengu, 420 bp (–300 to +120 of tengu); Pamp, 400 bp (–200 to +200 of amp). White arrowheads indicate the positions of transcripts from each template.

Mentions: To investigate the OY-M genes regulated by RpoDOY, we performed in vitro transcription assays using other templates. Many other bacteria housekeeping genes have sigma 70-type promoters26272829, so we used the upstream regions of four housekeeping genes of OY-M [the protein chain initiation factor IF-3 (infC), 50S ribosomal subunit protein L13 (rplM), 30S ribosomal subunit protein S4 (rpsD), and RNA polymerase sigma70 factor (rpoD) genes] for in vitro transcription as templates (PinfC, PrplM, PrpsD, and PrpoD, respectively). In addition, the upstream region of the molecular chaperone gene (ibpA) was used as a template (PibpA) because a previous study suggested that it is regulated by RpoD in an AT-rich bacterium ‘Ca. Blochmannia floridanus’30, while in some other bacteria, ibpA is reported to be regulated by an alternative heat shock sigma factor RpoH3132. Our in vitro transcription assays revealed that RNAPEc–RpoDOY produced specific transcripts from the four templates (PinfC, PrplM, PrpsD, and PibpA), but did not produce a transcript from PrpoD (Fig. 3). When only RNAPEc, which lacks RpoDOY, was added to the reaction, no specific transcripts were observed from any of these templates (Fig. 3). These results indicate that RpoDOY mediates the transcription of many phytoplasma housekeeping genes, although with some exceptions. Considering the previous findings that RNAP containing the principal sigma factor transcribes the majority of the housekeeping genes3, RpoDOY is likely to play a role as the principal sigma factor.


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)

In vitro transcription with templates containing the promoter region of various gene categories.RNAPEc-RpoDOY and DNA templates were incubated with NTP, including [γ-32P]CTP in the absence (–) or presence (+) of RpoDOY. The length of each template is as follows: PibpA, 713 bp (–512 to +201 of ibpA); PinfC, 702 bp (–500 to +202 of infC); PrplM, 700 bp (–500 to +200 of rplM); PrpsD, 650 bp (–450 to +200 of rpsD); PrpoD, 624 bp (–400 to +224 of rpoD); P157, 500 bp (–300 to +200 of PAM157); P289, 500 bp (–500 to –1 of PAM289); P486, 566 bp (–300 to +266 of PAM486); Ptengu, 420 bp (–300 to +120 of tengu); Pamp, 400 bp (–200 to +200 of amp). White arrowheads indicate the positions of transcripts from each template.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: In vitro transcription with templates containing the promoter region of various gene categories.RNAPEc-RpoDOY and DNA templates were incubated with NTP, including [γ-32P]CTP in the absence (–) or presence (+) of RpoDOY. The length of each template is as follows: PibpA, 713 bp (–512 to +201 of ibpA); PinfC, 702 bp (–500 to +202 of infC); PrplM, 700 bp (–500 to +200 of rplM); PrpsD, 650 bp (–450 to +200 of rpsD); PrpoD, 624 bp (–400 to +224 of rpoD); P157, 500 bp (–300 to +200 of PAM157); P289, 500 bp (–500 to –1 of PAM289); P486, 566 bp (–300 to +266 of PAM486); Ptengu, 420 bp (–300 to +120 of tengu); Pamp, 400 bp (–200 to +200 of amp). White arrowheads indicate the positions of transcripts from each template.
Mentions: To investigate the OY-M genes regulated by RpoDOY, we performed in vitro transcription assays using other templates. Many other bacteria housekeeping genes have sigma 70-type promoters26272829, so we used the upstream regions of four housekeeping genes of OY-M [the protein chain initiation factor IF-3 (infC), 50S ribosomal subunit protein L13 (rplM), 30S ribosomal subunit protein S4 (rpsD), and RNA polymerase sigma70 factor (rpoD) genes] for in vitro transcription as templates (PinfC, PrplM, PrpsD, and PrpoD, respectively). In addition, the upstream region of the molecular chaperone gene (ibpA) was used as a template (PibpA) because a previous study suggested that it is regulated by RpoD in an AT-rich bacterium ‘Ca. Blochmannia floridanus’30, while in some other bacteria, ibpA is reported to be regulated by an alternative heat shock sigma factor RpoH3132. Our in vitro transcription assays revealed that RNAPEc–RpoDOY produced specific transcripts from the four templates (PinfC, PrplM, PrpsD, and PibpA), but did not produce a transcript from PrpoD (Fig. 3). When only RNAPEc, which lacks RpoDOY, was added to the reaction, no specific transcripts were observed from any of these templates (Fig. 3). These results indicate that RpoDOY mediates the transcription of many phytoplasma housekeeping genes, although with some exceptions. Considering the previous findings that RNAP containing the principal sigma factor transcribes the majority of the housekeeping genes3, RpoDOY is likely to play a role as the principal sigma factor.

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