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To survive and proliferate, bacteria have to respond to a plethora of fluctuating signals within their habitats. Transcriptional control is one crucial entry point for such signal-responsive adaption responses. In this thesis I present new insights into the signal-responsive control of two specific transcriptional regulators that belong to a specialized class of mechano-transcriptional regulators. These regulators employ ATP-hydrolysis to engage and remodel σ54-RNA polymerase, which allows transcriptional initiation from the promoters they control. In the first part of my thesis I present findings on DmpR – the obligate activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR is a sensory-regulator that can only transition to its active multimeric form upon binding a phenolic compound and ATP. Previous work has established that binding of phenolic effectors by the N-terminal domain of DmpR relieves inter-domain repression of its central ATPase domain and further that a structured inter-domain linker between the phenolic- and ATP-binding domains is involved in coupling these processes. However, the mechanism underlying this coupling remained enigmatic. Here I present evidence that a tyrosine residue of the inter-domain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and phenolic-responsive transcriptional activation by DmpR. A model is presented in which binding of phenolics relocates Y233 from the ATP-binding site to synchronise signal-reception with multimerisation to provide appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand-responsive mechano-transcriptional regulators, the model is likely to be pertinent for numerous members of this family. The finding that an alanine substitution of Y233 enhances transcriptional responses adds a new approach to manipulating the sensitivity of this class of proteins and thereby generate hyper-sensitive detectors of aromatic

To survive and proliferate, bacteria have to respond to a plethora of fluctuating signals within their habitats. Transcriptional control is one crucial entry point for such signal-responsive adaption responses. In this thesis I present new insights into the signal-responsive control of two specific transcriptional regulators that belong to a specialized class of mechano-transcriptional regulators. These regulators employ ATP-hydrolysis to engage and remodel σ54-RNA polymerase, which allows transcriptional initiation from the promoters they control. In the first part of my thesis I present findings on DmpR – the obligate activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR is a sensory-regulator that can only transition to its active multimeric form upon binding a phenolic compound and ATP. Previous work has established that binding of phenolic effectors by the N-terminal domain of DmpR relieves inter-domain repression of its central ATPase domain and further that a structured inter-domain linker between the phenolic- and ATP-binding domains is involved in coupling these processes. However, the mechanism underlying this coupling remained enigmatic. Here I present evidence that a tyrosine residue of the inter-domain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and phenolic-responsive transcriptional activation by DmpR. A model is presented in which binding of phenolics relocates Y233 from the ATP-binding site to synchronise signal-reception with multimerisation to provide appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand-responsive mechano-transcriptional regulators, the model is likely to be pertinent for numerous members of this family. The finding that an alanine substitution of Y233 enhances transcriptional responses adds a new approach to manipulating the sensitivity of this class of proteins and thereby generate hyper-sensitive detectors of aromatic

To survive and proliferate, bacteria have to respond to a plethora of fluctuating signals within their habitats. Transcriptional control is one crucial entry point for such signal-responsive adaption responses. In this thesis I present new insights into the signal-responsive control of two specific transcriptional regulators that belong to a specialized class of mechano-transcriptional regulators. These regulators employ ATP-hydrolysis to engage and remodel σ54-RNA polymerase, which allows transcriptional initiation from the promoters they control. In the first part of my thesis I present findings on DmpR – the obligate activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR is a sensory-regulator that can only transition to its active multimeric form upon binding a phenolic compound and ATP. Previous work has established that binding of phenolic effectors by the N-terminal domain of DmpR relieves inter-domain repression of its central ATPase domain and further that a structured inter-domain linker between the phenolic- and ATP-binding domains is involved in coupling these processes. However, the mechanism underlying this coupling remained enigmatic. Here I present evidence that a tyrosine residue of the inter-domain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and phenolic-responsive transcriptional activation by DmpR. A model is presented in which binding of phenolics relocates Y233 from the ATP-binding site to synchronise signal-reception with multimerisation to provide appropriate sensitivity of the transcriptional response. Given that Y233 counterparts are present in many ligand-responsive mechano-transcriptional regulators, the model is likely to be pertinent for numerous members of this family. The finding that an alanine substitution of Y233 enhances transcriptional responses adds a new approach to manipulating the sensitivity of this class of proteins and thereby generate hyper-sensitive detectors of aromatic

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence fo

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence fo

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence fo

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence fo

Metabolically versatile bacteria have evolved diverse strategies to adapt to different environmental niches and respond to fluctuating physico-chemical parameters. In order to survive in soil and water habitats, they employ specific and global regulatory circuits to integrate external and internal signals to counteract stress and optimise their energy status. One strategic endurance mechanism is the ability to choose the most energetically favourable carbon source amongst a number on offer. Pseudomonas putida strains possess large genomes that underlie much of their ability to use diverse carbon sources as growth substrates. Their metabolic potential is frequently expanded by possession of catabolic plasmids to include the ability to grow at the expense of seemingly obnoxious carbon sources such as phenols. However, this ability comes with a metabolic price tag. Carbon source repression is one of the main regulatory networks employed to subvert use of these expensive pathways in favour of alternative sources that provide a higher metabolic gain. This thesis identifies some of the key regulatory elements and factors used by P. putida to supress expression of plasmid-encoded enzymes for degradation of phenols until they are beneficial. I first present evidence for a newly identified DNA and RNA motif within the regulatory region of the gene encoding the master regulator of phenol catabolism – DmpR. The former of these motifs functions to decrease the number of transcripts originating from the dmpR promoter, while the latter mediates a regulatory checkpoint for translational repression by Crc – the carbon repression control protein of P. putida. The ability of Crc to form repressive riboprotein complexes with RNA is shown to be dependent on the RNA chaperone protein Hfq – a co-partnership demonstrated to be required for many previously identified Crc-targets implicated in hierarchical assimilation of different carbon sources in P. putida. Finally, I present evidence fo

Highly sensitive calorimetry was evaluated for its potential for the monitoring of the bacterial contamination of drinking water. For that purpose, water samples were added to different media and bacterial growth was followed microcalorimetrically. Drinking water samples were either tested untreated or after artificial contamination with selected bacterial strains. Two bacteria, two growth media and two growth conditions (i.e. aerobic and anaerobic) were applied. Even minor initial bacterial contamination (1–100 cells mL−1) gave rise to calorimetric signals after 5–17 h growth. Calorimetry was thus faster than detection of bacterial contamination by plating on agar and inspection of colony formation, which was performed for comparison. It was further demonstrated theoretically that calorimetric detection is superior to colony detection. The heat production curves were characteristic for the strain, the medium and the growth conditions. It is hypothesized that a further refinement of the microcalorimetric method via the application of sets of specific media in combination with selective growth conditions should allow delimiting the identity of the contaminant.

SCOPUS: le.j, info:eu-repo/semantics/published

The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to

The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to

The σ54-dependent Po promoter drives transcription of an operon that encodes a suite of enzymes for (methyl)phenols catabolism. Transcription from Po is controlled by the sensor-activator DmpR that binds (methyl)phenol effectors to take up its active form. The σ54 factor imposes kinetic constraints on transcriptional initiation by the σ54-RNA polymerase holoenzyme which cannot undergo transition from the closed complex without the aid of the activator. DmpR acts from a distance on promoter-bound σ54-holoenzyme, and physical contact between the two players is facilitated by the DNA-bending protein IHF. The bacterial alarmone ppGpp and DksA directly bind RNA polymerase to have far reaching consequences on global transcriptional capacity in the cell. The work presented in this thesis uses the DmpR-regulated Po promoter as a framework to dissect how these two regulatory molecules act in vivo to control the functioning of σ54-dependent transcription. The strategies employed involved development of i) a series of hybrid σ54-promoters that could be directly compared and in which key DNA elements could be manipulated ii) mutants incapable of synthesizing ppGpp and/or DksA, iii) reconstituted in vitro transcription systems, and iv) genetic selection and purification of mutant RNA polymerases that bypass the need for ppGpp and DksA in vivo. The collective results presented show that the effects of ppGpp and DksA on σ54-dependent transcription are major, with simultaneous loss of these regulatory molecules essentially abolishing σ54-transcription in intact cells. However, neither of these regulatory molecules have discernable effects on in vitro reconstituted σ54-transcription, suggesting an indirect mechanism of control. The major effects of ppGpp and DksA in vivo cannot be accounted for by consequent changes in the levels of DmpR or other specific proteins needed for σ54-transcription. The data presented here shows i) that the effects of loss of ppGpp and DksA are related to