Eleonore Skärfstad

The role of the alarmone (p)ppGpp in sigma N competition for core RNA polymerase.

Some promoters, including the DmpR-controlled ςN-dependent Po promoter, are effectively rendered silent in cells lacking the nutritional alarmone (p)ppGpp. Here we demonstrate that four mutations within the housekeeping ςD-factor can restore ςN-dependent Po transcription in the absence of (p)ppGpp. Using both in vitro and in vivotranscription competition assays, we show that all the four ςD mutant proteins are defective in their ability to compete with ςN for available core RNA polymerase and that the magnitude of the defect reflects the hierarchy of restoration of transcription from Po in (p)ppGpp-deficient cells. Consistently, underproduction of ςD or overproduction of the anti-ςD protein Rsd were also found to allow (p)ppGpp-independent transcription from the ςN-Po promoter. Together with data from the direct effects of (p)ppGpp on ςN-dependent Po transcription and ς-factor competition, the results support a model in which (p)ppGpp serves as a master global regulator of transcription by differentially modulating alternative ς-factor competition to adapt to changing cellular nutritional demands.

The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ54-dependent transcription

The RNA polymerase‐binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)‐responsive control of transcription from σ70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivoσ54 transcription even though they do not have any major direct effects on σ54 transcription in reconstituted in vitro transcription and σ‐factor competition assays, (ii) that previously defined mutations rendering the housekeeping σ70 less effective at competing with σ54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from σ54 promoters in vivo reflects the innate affinity of the promoters for σ54‐RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of σ54‐dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated σ70 promoters.

Metabolism‐dependent taxis towards (methyl)phenols is coupled through the most abundant of three polar localized Aer‐like proteins of Pseudomonas putida

Comparatively little is known about directed motility of environmental bacteria to common aromatic pollutants. Here, by expressing different parts of a (methyl)phenol-degradative pathway and the use of specific mutants, we show that taxis of Pseudomonas putida towards (methyl)phenols is dictated by its ability to catabolize the aromatic compound. Thus, in contrast to previously described chemoreceptor-mediated chemotaxis mechanisms towards benzoate, naphthalene and toluene, taxis in response to (methyl)phenols is mediated by metabolism-dependent behaviour. Here we show that P. putida differentially expresses three Aer-like receptors that are all polar-localized through interactions with CheA, and that inactivation of the most abundant Aer2 protein significantly decreases taxis towards phenolics. In addition, the participation of a sensory signal transduction protein composed of a PAS, a GGDEF and an EAL domain in motility towards these compounds is demonstrated. The results are discussed in the context of the versatility of metabolism-dependent coupling and the necessity for P. putida to integrate diverse metabolic signals from its native heterogeneous soil and water environments.

Properties of RNA Polymerase Bypass Mutants IMPLICATIONS FOR THE ROLE OF ppGpp AND ITS CO-FACTOR DksA IN CONTROLLING TRANSCRIPTION DEPENDENT ON σ54

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain σ70-dependent promoters. A number of promoters that are dependent on alternative σ-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived σ54-dependent Po promoter and several other σ54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro σ54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the β- and β′-subunits of core RNA polymerase that allow high levels of transcription from the σ54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with σ-factors to favor σ54-holoenzyme formation over that with σ70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent σ70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of σ54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent σ70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative σ-factors is controlled to meet cellular requirements.

Role of the DmpR-Mediated Regulatory Circuit in Bacterial Biodegradation Properties in Methylphenol-Amended Soils

ABSTRACT Pathway substrates and some structural analogues directly activate the regulatory protein DmpR to promote transcription of thedmp operon genes encoding the (methyl)phenol degradative pathway of Pseudomonas sp. strain CF600. While a wide range of phenols can activate DmpR, the location and nature of substituents on the basic phenolic ring can limit the level of activation and thus utilization of some compounds as assessed by growth on plates. Here we address the role of the aromatic effector response of DmpR in determining degradative properties in two soil matrices that provide different nutritional conditions. Using the wild-type system and an isogenic counterpart containing a DmpR mutant with enhanced ability to respond to para-substituted phenols, we demonstrate (i) that the enhanced in vitro biodegradative capacity of the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to 4-methylphenol-contaminated soil led to rapid selection of a subpopulation exhibiting enhanced capacities to degrade the compound. Genetic and functional analyses of 10 of these derivatives demonstrated that all harbored a single mutation in the sensory domain of DmpR that mediated the phenotype in each case. These findings establish a dominating role for the aromatic effector response of DmpR in determining degradation properties. Moreover, the results indicate that the ability to rapidly adapt regulator properties to different profiles of polluting compounds may underlie the evolutionary success of DmpR-like regulators in controlling aromatic catabolic pathways.

σ54-Promoter Discrimination and Regulation by ppGpp and DksA

The σ54-factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of σ54-dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of σ54-dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of σ54-dependent transcription in combination with a series of σ54-promoters with graded affinities for σ54-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted σ54-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous σ54-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive σ54-transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity σ54-promoters that is conferred by the strong DNA binding properties of the P. putida σ54-RNA polymerase holoenzyme.

The σ-factor FliA, ppGpp and DksA coordinate transcriptional control of the aer2 gene of Pseudomonas putida

Here the sigma-factor requirement for transcription of three similar, but differentially regulated, aer genes of Pseudomonas putida KT2440 is investigated. Previous work has shown that the three Aer proteins, like chemoreceptors, colocalize to a single pole in a CheA-dependent manner. Lack of Aer2 - the most abundant of these three proteins - mediates defects in metabolism-dependent taxis and aerotaxis, while lack of Aer1 or Aer3 has no apparent phenotype. We show, using wild-type and mutant P. putida derivatives combined with P. putida reconstituted FliA- (sigma(28)) and sigma(70)-dependent in vitro transcription assays, that transcription of aer2 is coupled to motility through the flagella sigma-factor FliA, while sigma(70) is responsible for transcription of aer1 and aer3. By comparing activities of the wild-type and mutant forms of the aer2 promoter, we present evidence (i) that transcription from FliA-dependent Paer2 is enhanced by changes towards the Escherichia coli consensus for FliA promoters rather than towards that of P. putida, (ii) that the nature of the AT-rich upstream region is important for both output and sigma(70) discrimination of this promoter, and (iii) that Paer2 output is directly stimulated by the bacterial alarmone ppGpp and its cofactor DksA.

A hyper-mutant of the unusual σ70-Pr promoter bypasses synergistic ppGpp/DksA co-stimulation

The activities of promoters can be temporally and conditionally regulated by mechanisms other than classical DNA-binding repressors and activators. One example is the inherently weak σ70-dependent Pr promoter that ultimately controls catabolism of phenolic compounds. The activity of Pr is up-regulated through the joint action of ppGpp and DksA that enhance the performance of RNA polymerase at this promoter. Here, we report a mutagenesis analysis that revealed substantial differences between Pr and other ppGpp/DksA co-stimulated promoters. In vitro transcription and RNA polymerase binding assays show that it is the T at the −11 position of the extremely suboptimal −10 element of Pr that underlies both poor binding of σ70-RNAP and a slow rate of open complex formation—the process that is accelerated by ppGpp and DksA. Our findings support the idea that collaborative action of ppGpp and DksA lowers the rate-limiting transition energy required for conversion between intermediates on the road to open complex formation.

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

The Pr promoter is the first verified member of a class of bacterial σ70-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 σ70-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.

Characterization of the transcriptional stimulatory properties of the Pseudomonas putida RapA protein

RNA polymerase-associated factors can significantly affect its performance at specific promoters. Here we identified a Pseudomonas putida RNA polymerases-associated protein as a homolog of Escherichia coli RapA. We found that P. putida RapA stimulates the transcription from promoters dependent on a variety of σ-factors (σ(70), σ(S), σ(54), σ(32), σ(E)) in vitro. The level of stimulation varied from 2- to 10-fold, with the maximal effect observed with the σ(E)-dependent PhtrA promoter. Stimulation by RapA was apparent in the multi-round reactions and was modulated by salt concentration in vitro. However, in contrast to findings with E. coli RapA, P. putida RapA-mediated stimulation of transcription was also evident using linear templates. These properties of P. putida RapA were apparent using either E. coli- or P. putida-derived RNA polymerases. Analysis of individual steps of transcription revealed that P. putida RapA enhances the stability of competitor-resistant open-complexes formed by RNA polymerase at promoters. In vivo, P. putida RapA can complement the inhibitory effect of high salt on growth of an E. coli RapA null strain. However, a P. putida RapA null mutant was not sensitive to high salt. The in vivo effects of lack of RapA were only detectable for the σ(E)-PhtrA promoter where the RapA-deficiency resulted in lower activity. The presented characteristics of P. putida RapA indicate that its functions may extend beyond a role in facilitating RNA polymerase recycling to include a role in transcription initiation efficiency.