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Elucidation of sigma factor-associated networks in Pseudomonas aeruginosa reveals a modular architecture with limited and function-specific crosstalk.

Schulz S, Eckweiler D, Bielecka A, Nicolai T, Franke R, Dötsch A, Hornischer K, Bruchmann S, Düvel J, Häussler S - PLoS Pathog. (2015)

Bottom Line: We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS).Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes.Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bacteriology, TWINCORE GmbH, Center for Clinical and Experimental Infection Research, a joint venture of the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover, Germany; Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

ABSTRACT
Sigma factors are essential global regulators of transcription initiation in bacteria which confer promoter recognition specificity to the RNA polymerase core enzyme. They provide effective mechanisms for simultaneously regulating expression of large numbers of genes in response to challenging conditions, and their presence has been linked to bacterial virulence and pathogenicity. In this study, we constructed nine his-tagged sigma factor expressing and/or deletion mutant strains in the opportunistic pathogen Pseudomonas aeruginosa. To uncover the direct and indirect sigma factor regulons, we performed mRNA profiling, as well as chromatin immunoprecipitation coupled to high-throughput sequencing. We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS). Our integrated approach revealed a highly modular network architecture which is composed of insulated functional sigma factor modules. Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes. Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.

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Related in: MedlinePlus

Functional characterization of primary sigma factor networks.The PseudoCAP annotation [55] was used to categorize the members of the sigma factor networks (Fig. 3, S6 Table). For each category the enrichment factor based on the prevalence of the specified class in the regulon compared to the whole genome was calculated. The P values of the enriched categories are provided in S8 Table.
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ppat.1004744.g004: Functional characterization of primary sigma factor networks.The PseudoCAP annotation [55] was used to categorize the members of the sigma factor networks (Fig. 3, S6 Table). For each category the enrichment factor based on the prevalence of the specified class in the regulon compared to the whole genome was calculated. The P values of the enriched categories are provided in S8 Table.

Mentions: Of note, meeting two out of the three of the criteria to define the sigma factor regulons decreased the regulon size of e.g. RpoH from 268 when meeting just one of the criteria (RNA–seq) (Table 1) to 96 (Table 2). However, on the other hand the regulon sizes of RpoN, AlgU and RpoS even increased, indicating that ChIP-seq in combination with a motif scan uncovered additional sigma factor binding sites. The validity of the selection criteria was further verified by functionally categorizing the members of each primary sigma factor regulon by the use of the PseudoCAP annotation [55]. The results are summarized in Fig. 4 (the enrichment values and their P values are listed in S8 Table). As expected, the AlgU regulon comprises genes of alginate biosynthesis and cellular homeostasis [59–63], the motility sigma factor FliA influences genes involved in chemotaxis and motility [64,65] and PvdS directs the pyoverdine biosynthesis genes [66] which are assigned to the category adaptation/protection. The heat-shock sigma factor RpoH governs the gene expression of chaperones and heat-shock proteins [67], while RpoN controls genes of nitrogen metabolism, chemotaxis, motility and attachment [68–70]. The stationary phase sigma factor RpoS regulates quorum sensing genes as well as genes involved in general adaptation processes [71,72]. A more detailed description of the individual regulons is provided in the supplementary material (S1 Text).


Elucidation of sigma factor-associated networks in Pseudomonas aeruginosa reveals a modular architecture with limited and function-specific crosstalk.

Schulz S, Eckweiler D, Bielecka A, Nicolai T, Franke R, Dötsch A, Hornischer K, Bruchmann S, Düvel J, Häussler S - PLoS Pathog. (2015)

Functional characterization of primary sigma factor networks.The PseudoCAP annotation [55] was used to categorize the members of the sigma factor networks (Fig. 3, S6 Table). For each category the enrichment factor based on the prevalence of the specified class in the regulon compared to the whole genome was calculated. The P values of the enriched categories are provided in S8 Table.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362757&req=5

ppat.1004744.g004: Functional characterization of primary sigma factor networks.The PseudoCAP annotation [55] was used to categorize the members of the sigma factor networks (Fig. 3, S6 Table). For each category the enrichment factor based on the prevalence of the specified class in the regulon compared to the whole genome was calculated. The P values of the enriched categories are provided in S8 Table.
Mentions: Of note, meeting two out of the three of the criteria to define the sigma factor regulons decreased the regulon size of e.g. RpoH from 268 when meeting just one of the criteria (RNA–seq) (Table 1) to 96 (Table 2). However, on the other hand the regulon sizes of RpoN, AlgU and RpoS even increased, indicating that ChIP-seq in combination with a motif scan uncovered additional sigma factor binding sites. The validity of the selection criteria was further verified by functionally categorizing the members of each primary sigma factor regulon by the use of the PseudoCAP annotation [55]. The results are summarized in Fig. 4 (the enrichment values and their P values are listed in S8 Table). As expected, the AlgU regulon comprises genes of alginate biosynthesis and cellular homeostasis [59–63], the motility sigma factor FliA influences genes involved in chemotaxis and motility [64,65] and PvdS directs the pyoverdine biosynthesis genes [66] which are assigned to the category adaptation/protection. The heat-shock sigma factor RpoH governs the gene expression of chaperones and heat-shock proteins [67], while RpoN controls genes of nitrogen metabolism, chemotaxis, motility and attachment [68–70]. The stationary phase sigma factor RpoS regulates quorum sensing genes as well as genes involved in general adaptation processes [71,72]. A more detailed description of the individual regulons is provided in the supplementary material (S1 Text).

Bottom Line: We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS).Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes.Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bacteriology, TWINCORE GmbH, Center for Clinical and Experimental Infection Research, a joint venture of the Hannover Medical School and the Helmholtz Centre for Infection Research, Hannover, Germany; Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany.

ABSTRACT
Sigma factors are essential global regulators of transcription initiation in bacteria which confer promoter recognition specificity to the RNA polymerase core enzyme. They provide effective mechanisms for simultaneously regulating expression of large numbers of genes in response to challenging conditions, and their presence has been linked to bacterial virulence and pathogenicity. In this study, we constructed nine his-tagged sigma factor expressing and/or deletion mutant strains in the opportunistic pathogen Pseudomonas aeruginosa. To uncover the direct and indirect sigma factor regulons, we performed mRNA profiling, as well as chromatin immunoprecipitation coupled to high-throughput sequencing. We furthermore elucidated the de novo binding motif of each sigma factor, and validated the RNA- and ChIP-seq results by global motif searches in the proximity of transcriptional start sites (TSS). Our integrated approach revealed a highly modular network architecture which is composed of insulated functional sigma factor modules. Analysis of the interconnectivity of the various sigma factor networks uncovered a limited, but highly function-specific, crosstalk which orchestrates complex cellular processes. Our data indicate that the modular structure of sigma factor networks enables P. aeruginosa to function adequately in its environment and at the same time is exploited to build up higher-level functions by specific interconnections that are dominated by a participation of RpoN.

Show MeSH
Related in: MedlinePlus