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Pr is a member of a restricted class of σ70-dependent promoters that lack a recognizable -10 element.

Del Peso-Santos T, Landfors M, Skärfstad E, Ryden P, Shingler V - Nucleic Acids Res. (2012)

Bottom Line: We found that highly similar promoters control plasmid- and chromosomally- encoded phenol degradative systems in various Pseudomonads.However, using a purpose-designed promoter-search algorithm and activity analysis of potential candidate promoters, no bona fide Pr-like promoter could be found in the entire genome of P. putida KT2440.Hence, Pr-like σ(70)-promoters, which have the potential to be a widely distributed class of previously unrecognized promoters, are in fact highly restricted and remain in a class of their own.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå SE 90187, Sweden.

ABSTRACT
The Pr promoter is the first verified member of a class of bacterial σ(70)-promoters that only possess a single match to consensus within its -10 element. In its native context, the activity of this promoter determines the ability of Pseudomonas putida CF600 to degrade phenolic compounds, which provides proof-of-principle for the significance of such promoters. Lack of identity within the -10 element leads to non-detection of Pr-like promoters by current search engines, because of their bias for detection of the -10 motif. Here, we report a mutagenesis analysis of Pr that reveals strict sequence requirements for its activity that includes an essential -15 element and preservation of non-consensus bases within its -35 and -10 elements. We found that highly similar promoters control plasmid- and chromosomally- encoded phenol degradative systems in various Pseudomonads. However, using a purpose-designed promoter-search algorithm and activity analysis of potential candidate promoters, no bona fide Pr-like promoter could be found in the entire genome of P. putida KT2440. Hence, Pr-like σ(70)-promoters, which have the potential to be a widely distributed class of previously unrecognized promoters, are in fact highly restricted and remain in a class of their own.

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Related in: MedlinePlus

Analysis of the −15 element of Pr. The graph shows the stationary phase luciferase activities of E. coli DH5 harbouring −38 to +8 Pr luciferase transcriptional reporter plasmid variants encompassing different mutations in the −10 and/or −15 elements. The plasmid names and sequences of these −38 to +8 Pr variants are shown to the left. The −35 and short −10 elements are underlined, whereas bases matching the consensus −35, −15 and −10 elements are shown in red bold-case letters. Substitutions in the sequence that differ from the consensus are indicated in black and bold-case letters as is the +1. Values for relative transcription are from duplicate determinations in two to four independent experiments, normalized by setting the value of Pr WT as 100% activity. Error bars are ±SD.
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gks934-F5: Analysis of the −15 element of Pr. The graph shows the stationary phase luciferase activities of E. coli DH5 harbouring −38 to +8 Pr luciferase transcriptional reporter plasmid variants encompassing different mutations in the −10 and/or −15 elements. The plasmid names and sequences of these −38 to +8 Pr variants are shown to the left. The −35 and short −10 elements are underlined, whereas bases matching the consensus −35, −15 and −10 elements are shown in red bold-case letters. Substitutions in the sequence that differ from the consensus are indicated in black and bold-case letters as is the +1. Values for relative transcription are from duplicate determinations in two to four independent experiments, normalized by setting the value of Pr WT as 100% activity. Error bars are ±SD.

Mentions: The Pr promoter possesses two out of four matches, −14G and −13G, to the consensus −15 element sequence (−15AGGC−12, consensus −15TGGT−12). To determine if this potential −15 element plays an important role in Pr activity, we constructed additional transcriptional reporter plasmids carrying derivatives of Pr (−38 to +8) controlling the luxAB genes and assayed promoter activity (Figure 5). Simultaneous substitution of both these residues completely abolished detectable Pr activity (G−14A and G−13A in pVI2263, Figure 5). Individual analysis of the contributions of the −14G and −13G bases showed that both residues are involved in maintaining wild-type Pr activity. Substitution of the G at −13 position for any other base resulted in greatly decreased promoter activity as compared with wild type (25% for G−13C, 7.5% for G−13A and 11.4% for G−13T). Similarly, substitution of the G in the −14 position to either A or C also resulted in greatly reduced promoter activity (to ∼11–12% of wild-type Pr), whereas the G−14T substitution retained 62% promoter activity. Examination of the sequence resulting from the G−14T substitution suggests that the promoter activity observed might be due to creation of a new promoter comprising an almost perfect −15 element and a non-existing short −10 (−15TGCT−12/−11GGCTA−7, consensus −15TGGT12/−11ATAAT−7) spaced 18 bp from the −35 element of Pr. If this was indeed the case, it would imply that a promoter can be functional with only a −35 and −15 element. However, we found that Pr mutant derivatives that improve the −15 element but destroy any match to the short −10 element do not show any promoter activity (compare variants pVI2202 with pVI2207, pVI2254 with pVI2264 and pVI2253 with pVI1020; Figure 5). In addition, a suboptimal 18-bp spacer region between the −35 and −10 elements renders Pr non-functional (Figure 1E). In the light of these results, we conclude that the 62% promoter activity retained by the Pr G−14T variant is because of the tolerance of a T at this position, rather than through creation of a new promoter.Figure 5.


Pr is a member of a restricted class of σ70-dependent promoters that lack a recognizable -10 element.

Del Peso-Santos T, Landfors M, Skärfstad E, Ryden P, Shingler V - Nucleic Acids Res. (2012)

Analysis of the −15 element of Pr. The graph shows the stationary phase luciferase activities of E. coli DH5 harbouring −38 to +8 Pr luciferase transcriptional reporter plasmid variants encompassing different mutations in the −10 and/or −15 elements. The plasmid names and sequences of these −38 to +8 Pr variants are shown to the left. The −35 and short −10 elements are underlined, whereas bases matching the consensus −35, −15 and −10 elements are shown in red bold-case letters. Substitutions in the sequence that differ from the consensus are indicated in black and bold-case letters as is the +1. Values for relative transcription are from duplicate determinations in two to four independent experiments, normalized by setting the value of Pr WT as 100% activity. Error bars are ±SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526299&req=5

gks934-F5: Analysis of the −15 element of Pr. The graph shows the stationary phase luciferase activities of E. coli DH5 harbouring −38 to +8 Pr luciferase transcriptional reporter plasmid variants encompassing different mutations in the −10 and/or −15 elements. The plasmid names and sequences of these −38 to +8 Pr variants are shown to the left. The −35 and short −10 elements are underlined, whereas bases matching the consensus −35, −15 and −10 elements are shown in red bold-case letters. Substitutions in the sequence that differ from the consensus are indicated in black and bold-case letters as is the +1. Values for relative transcription are from duplicate determinations in two to four independent experiments, normalized by setting the value of Pr WT as 100% activity. Error bars are ±SD.
Mentions: The Pr promoter possesses two out of four matches, −14G and −13G, to the consensus −15 element sequence (−15AGGC−12, consensus −15TGGT−12). To determine if this potential −15 element plays an important role in Pr activity, we constructed additional transcriptional reporter plasmids carrying derivatives of Pr (−38 to +8) controlling the luxAB genes and assayed promoter activity (Figure 5). Simultaneous substitution of both these residues completely abolished detectable Pr activity (G−14A and G−13A in pVI2263, Figure 5). Individual analysis of the contributions of the −14G and −13G bases showed that both residues are involved in maintaining wild-type Pr activity. Substitution of the G at −13 position for any other base resulted in greatly decreased promoter activity as compared with wild type (25% for G−13C, 7.5% for G−13A and 11.4% for G−13T). Similarly, substitution of the G in the −14 position to either A or C also resulted in greatly reduced promoter activity (to ∼11–12% of wild-type Pr), whereas the G−14T substitution retained 62% promoter activity. Examination of the sequence resulting from the G−14T substitution suggests that the promoter activity observed might be due to creation of a new promoter comprising an almost perfect −15 element and a non-existing short −10 (−15TGCT−12/−11GGCTA−7, consensus −15TGGT12/−11ATAAT−7) spaced 18 bp from the −35 element of Pr. If this was indeed the case, it would imply that a promoter can be functional with only a −35 and −15 element. However, we found that Pr mutant derivatives that improve the −15 element but destroy any match to the short −10 element do not show any promoter activity (compare variants pVI2202 with pVI2207, pVI2254 with pVI2264 and pVI2253 with pVI1020; Figure 5). In addition, a suboptimal 18-bp spacer region between the −35 and −10 elements renders Pr non-functional (Figure 1E). In the light of these results, we conclude that the 62% promoter activity retained by the Pr G−14T variant is because of the tolerance of a T at this position, rather than through creation of a new promoter.Figure 5.

Bottom Line: We found that highly similar promoters control plasmid- and chromosomally- encoded phenol degradative systems in various Pseudomonads.However, using a purpose-designed promoter-search algorithm and activity analysis of potential candidate promoters, no bona fide Pr-like promoter could be found in the entire genome of P. putida KT2440.Hence, Pr-like σ(70)-promoters, which have the potential to be a widely distributed class of previously unrecognized promoters, are in fact highly restricted and remain in a class of their own.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå University, Umeå SE 90187, Sweden.

ABSTRACT
The Pr promoter is the first verified member of a class of bacterial σ(70)-promoters that only possess a single match to consensus within its -10 element. In its native context, the activity of this promoter determines the ability of Pseudomonas putida CF600 to degrade phenolic compounds, which provides proof-of-principle for the significance of such promoters. Lack of identity within the -10 element leads to non-detection of Pr-like promoters by current search engines, because of their bias for detection of the -10 motif. Here, we report a mutagenesis analysis of Pr that reveals strict sequence requirements for its activity that includes an essential -15 element and preservation of non-consensus bases within its -35 and -10 elements. We found that highly similar promoters control plasmid- and chromosomally- encoded phenol degradative systems in various Pseudomonads. However, using a purpose-designed promoter-search algorithm and activity analysis of potential candidate promoters, no bona fide Pr-like promoter could be found in the entire genome of P. putida KT2440. Hence, Pr-like σ(70)-promoters, which have the potential to be a widely distributed class of previously unrecognized promoters, are in fact highly restricted and remain in a class of their own.

Show MeSH
Related in: MedlinePlus