Genome-wide study predicts promoter-G4 DNA motifs regulate selective functions in bacteria: radioresistance of D. radiodurans involves G4 DNA-mediated regulation

Abstract

A remarkable number of guanine-rich sequences with potential to adopt non-canonical secondary structures called G-quadruplexes (or G4 DNA) are found within gene promoters. Despite growing interest, regulatory role of quadruplex DNA motifs in intrinsic cellular function remains poorly understood. Herein, we asked whether occurrence of potential G4 (PG4) DNA in promoters is associated with specific function(s) in bacteria. Using a normalized promoter-PG4-content (PG4(P)) index we analysed >60,000 promoters in 19 well-annotated species for (a) function class(es) and (b) gene(s) with enriched PG4(P). Unexpectedly, PG4-associated functional classes were organism specific, suggesting that PG4 motifs may impart specific function to organisms. As a case study, we analysed radioresistance. Interestingly, unsupervised clustering using PG4(P) of 21 genes, crucial for radioresistance, grouped three radioresistant microorganisms including Deinococcus radiodurans. Based on these predictions we tested and found that in presence of nanomolar amounts of the intracellular quadruplex-binding ligand N-methyl mesoporphyrin (NMM), radioresistance of D. radiodurans was attenuated by ~60%. In addition, important components of the RecF recombinational repair pathway recA, recF, recO, recR and recQ genes were found to harbour promoter-PG4 motifs and were also down-regulated in presence of NMM. Together these results provide first evidence that radioresistance may involve G4 DNA-mediated regulation and support the rationale that promoter-PG4s influence selective functions.

Figure 1.

Structure of the guanine quadruplex motif. Hydrogen-bonded self-assembly of guanine bases stabilized by monovalent cations form tetrads (left) that make the core of the four-stranded structure. Intramolecular quadruplex motifs are made of guanine tetrads linked by three loops of variable nucleotide length comprised of any of the four bases A, T, G or C (right).

Figure 2.

Promoter PG4 motifs define distinct functional classes in organisms. (a) Scheme for quadruplex-occurrence analysis in function classes (left panel) and individual genes (right panel). The index PG4_P was used for normalized content of potential quadruplex-forming sequence within individual promoters. Enrichment of promoters with significant PG4_P in a functional class was computed using the mean of randomly expected number drawn from 1000 simulations as described in Materials and Methods section. (b) Functional classes with higher than expected number of promoters having significant presence of PG4 motifs in E. coli; classes at least two standard deviation above expected are denoted with asterisk. (c) Heat map representation of cluster showing enrichment (z-score) of genes with significant PG4_P in functional classes across different organisms; blank squares indicate no gene with higher than expected PG4_P was found. (d) Heat map of clustering showing promoters of individual genes that have significant PG4_P across organisms. Escherichia coli was chosen as the reference organism and orthologues of the E. coli gene were used to construct gene-groups across 19 other organisms. Gene-groups where significant PG4_P (z > 2.0) was observed in at least three organisms (in addition to E. coli) are shown; Aca, Acidobacterium capsulatum; Afe, Acidithiobacillus ferrooxidans (ATCC 53993); Afo, Acidimicrobium ferrooxidans; Afr, Acidithiobacillus ferrooxidans (ATCC 23270); Art, Arthrobacter sp.; Bpf, Bacillus pseudofirmus; Bsu, Bacillus subtilis; Cai, Catenulispora acidiphila; Cbu, Catenulispora burnetii; Ddr, Deinococcus deserti; Dra, Deinococcus radiodurans; Eco, Escherichia coli; Gox, Gluconobacter oxydans; Hpy, Helicobacter pylori; Kra, Kineococcus radiotolerans; Msl, Methylocella silvestris; Nph, Natronomonas pharaonis; Rru, Rhodospirillum rubrum; Sul, Sulfurihydrogenibium sp.

Figure 2.

Promoter PG4 motifs define distinct functional classes in organisms. (a) Scheme for quadruplex-occurrence analysis in function classes (left panel) and individual genes (right panel). The index PG4_P was used for normalized content of potential quadruplex-forming sequence within individual promoters. Enrichment of promoters with significant PG4_P in a functional class was computed using the mean of randomly expected number drawn from 1000 simulations as described in Materials and Methods section. (b) Functional classes with higher than expected number of promoters having significant presence of PG4 motifs in E. coli; classes at least two standard deviation above expected are denoted with asterisk. (c) Heat map representation of cluster showing enrichment (z-score) of genes with significant PG4_P in functional classes across different organisms; blank squares indicate no gene with higher than expected PG4_P was found. (d) Heat map of clustering showing promoters of individual genes that have significant PG4_P across organisms. Escherichia coli was chosen as the reference organism and orthologues of the E. coli gene were used to construct gene-groups across 19 other organisms. Gene-groups where significant PG4_P (z > 2.0) was observed in at least three organisms (in addition to E. coli) are shown; Aca, Acidobacterium capsulatum; Afe, Acidithiobacillus ferrooxidans (ATCC 53993); Afo, Acidimicrobium ferrooxidans; Afr, Acidithiobacillus ferrooxidans (ATCC 23270); Art, Arthrobacter sp.; Bpf, Bacillus pseudofirmus; Bsu, Bacillus subtilis; Cai, Catenulispora acidiphila; Cbu, Catenulispora burnetii; Ddr, Deinococcus deserti; Dra, Deinococcus radiodurans; Eco, Escherichia coli; Gox, Gluconobacter oxydans; Hpy, Helicobacter pylori; Kra, Kineococcus radiotolerans; Msl, Methylocella silvestris; Nph, Natronomonas pharaonis; Rru, Rhodospirillum rubrum; Sul, Sulfurihydrogenibium sp.

Figure 2.

Promoter PG4 motifs define distinct functional classes in organisms. (a) Scheme for quadruplex-occurrence analysis in function classes (left panel) and individual genes (right panel). The index PG4_P was used for normalized content of potential quadruplex-forming sequence within individual promoters. Enrichment of promoters with significant PG4_P in a functional class was computed using the mean of randomly expected number drawn from 1000 simulations as described in Materials and Methods section. (b) Functional classes with higher than expected number of promoters having significant presence of PG4 motifs in E. coli; classes at least two standard deviation above expected are denoted with asterisk. (c) Heat map representation of cluster showing enrichment (z-score) of genes with significant PG4_P in functional classes across different organisms; blank squares indicate no gene with higher than expected PG4_P was found. (d) Heat map of clustering showing promoters of individual genes that have significant PG4_P across organisms. Escherichia coli was chosen as the reference organism and orthologues of the E. coli gene were used to construct gene-groups across 19 other organisms. Gene-groups where significant PG4_P (z > 2.0) was observed in at least three organisms (in addition to E. coli) are shown; Aca, Acidobacterium capsulatum; Afe, Acidithiobacillus ferrooxidans (ATCC 53993); Afo, Acidimicrobium ferrooxidans; Afr, Acidithiobacillus ferrooxidans (ATCC 23270); Art, Arthrobacter sp.; Bpf, Bacillus pseudofirmus; Bsu, Bacillus subtilis; Cai, Catenulispora acidiphila; Cbu, Catenulispora burnetii; Ddr, Deinococcus deserti; Dra, Deinococcus radiodurans; Eco, Escherichia coli; Gox, Gluconobacter oxydans; Hpy, Helicobacter pylori; Kra, Kineococcus radiotolerans; Msl, Methylocella silvestris; Nph, Natronomonas pharaonis; Rru, Rhodospirillum rubrum; Sul, Sulfurihydrogenibium sp.

Figure 3.

PG4_P-based analysis independently clusters radiation resistance species. Heat map representation of cluster diagram showing promoters of individual genes involved in imparting radiation resistance. Cluster was drawn for 18 organisms based on PG4_P of genes, with E. coli as the reference organism.

Figure 4.

Quadruplex-binding ligands and radiation resistance. NMM attenuates growth of (a) D. radiodurans and (b) D. geothermalis, following exposure to gamma irradiation; D. radiodurans and D. geothermalis levels at 0 kGy were used to estimate relative survival following 24–90 h of post-irradiation recovery at 32°C in presence of 0 nM or 50 nM NMM (left panel) and MIX (right panel), respectively. Values are mean ± standard error of three independent experiments. (c) Scheme showing PG4 motif positions within putative regulatory region of the recA operon. (d) Expression levels of recA and 16S rRNA genes in presence/absence of NMM in response to gamma irradiation (upper panel); quantification of relative intensities of the recA and 16S rRNA RT-PCR bands with respect to 0 nM NMM at respective radiation doses (lower panel). (e) Scheme showing PG4 motif in putative promoters of respective opreons with recF, recO, recR and recQ genes. (f) Expression levels of recF, recO, recR, recQ and 16S rRNA genes in presence/absence of NMM in response to gamma irradiation (left panel); relative quantification of the recF, recO, recR, recQ and 16S rRNA RT-PCR results with respect to 0 nM NMM at respective radiation doses (right panel). All PCR assays were performed in triplicates and values are presented as mean ± standard error; * denotes P < 0.05.

Figure 4.

Quadruplex-binding ligands and radiation resistance. NMM attenuates growth of (a) D. radiodurans and (b) D. geothermalis, following exposure to gamma irradiation; D. radiodurans and D. geothermalis levels at 0 kGy were used to estimate relative survival following 24–90 h of post-irradiation recovery at 32°C in presence of 0 nM or 50 nM NMM (left panel) and MIX (right panel), respectively. Values are mean ± standard error of three independent experiments. (c) Scheme showing PG4 motif positions within putative regulatory region of the recA operon. (d) Expression levels of recA and 16S rRNA genes in presence/absence of NMM in response to gamma irradiation (upper panel); quantification of relative intensities of the recA and 16S rRNA RT-PCR bands with respect to 0 nM NMM at respective radiation doses (lower panel). (e) Scheme showing PG4 motif in putative promoters of respective opreons with recF, recO, recR and recQ genes. (f) Expression levels of recF, recO, recR, recQ and 16S rRNA genes in presence/absence of NMM in response to gamma irradiation (left panel); relative quantification of the recF, recO, recR, recQ and 16S rRNA RT-PCR results with respect to 0 nM NMM at respective radiation doses (right panel). All PCR assays were performed in triplicates and values are presented as mean ± standard error; * denotes P < 0.05.

Figure 4.

Quadruplex-binding ligands and radiation resistance. NMM attenuates growth of (a) D. radiodurans and (b) D. geothermalis, following exposure to gamma irradiation; D. radiodurans and D. geothermalis levels at 0 kGy were used to estimate relative survival following 24–90 h of post-irradiation recovery at 32°C in presence of 0 nM or 50 nM NMM (left panel) and MIX (right panel), respectively. Values are mean ± standard error of three independent experiments. (c) Scheme showing PG4 motif positions within putative regulatory region of the recA operon. (d) Expression levels of recA and 16S rRNA genes in presence/absence of NMM in response to gamma irradiation (upper panel); quantification of relative intensities of the recA and 16S rRNA RT-PCR bands with respect to 0 nM NMM at respective radiation doses (lower panel). (e) Scheme showing PG4 motif in putative promoters of respective opreons with recF, recO, recR and recQ genes. (f) Expression levels of recF, recO, recR, recQ and 16S rRNA genes in presence/absence of NMM in response to gamma irradiation (left panel); relative quantification of the recF, recO, recR, recQ and 16S rRNA RT-PCR results with respect to 0 nM NMM at respective radiation doses (right panel). All PCR assays were performed in triplicates and values are presented as mean ± standard error; * denotes P < 0.05.