Victoria Shingler

Signal sensing by sigma 54-dependent regulators : derepression as a control mechanism.

Transcription by RNA polymerase utilizing the alternative sigma factor σ54 is regulated by a distinct class of positive activators designated the σ54‐dependent family. The activities of these regulators are themselves modulated in response to a wide variety of environmental signals. Factors that modulate the expression or the activity of the regulatory protein in response to chemical and metabolic changes are ultimately responsible for determining the level of expression of σ54‐dependent genes and hence the diverse bacterial functions that they encode. Many members of the σ54‐dependent family are part of two‐component sensor‐response systems. This MicroReview emphasizes recent data concerning the activities of a distinct subgroup of the σ54‐dependent regulators that directly sense and respond with transcriptional activation to the presence of small effector molecules in their environment. The functional consequences of effector activation in terms of regulation of the enzymatic (ATPase) activity of these transcriptional activators and interdomain interactions are discussed.

Genetics and biochemistry of phenol degradation by Pseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.

Regulation of Alternative Sigma Factor Use

Alternative bacterial sigma factors bind the catalytic core RNA polymerase to confer promoter selectivity on the holoenzyme. The different holoenzymes are thus programmed to recognize the distinct promoter classes in the genome to allow coordinated activation of discrete sets of genes needed for adaptive responses. To form the holoenzymes, the different sigma factors must be available to compete for their common substrate (core RNA polymerase). This review highlights (a) the roles of antisigma factors in controlling the availability of alternative sigma factors and (b) the involvement of diverse regulatory molecules that promote the use of alternative sigma factors through subversion of the domineering housekeeping σ(70). The latter include the nucleotide alarmone ppGpp and small proteins (DksA, Rsd, and Crl), which directly target the transcriptional machinery to mediate their effects.

The alarmone (p)ppGpp mediates physiological-responsive control at the sigma 54-dependent Po promoter.

Transcription from the Pseudomonas‐derived σ54‐dependent Po promoter of the dmp operon is mediated by the aromatic‐responsive regulator DmpR. However, physiological control is superimposed on this regulatory system causing silencing of the DmpR‐mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached. Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo. Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non‐permissive conditions. Conversely (p)ppGpp‐deficient Escherichia coli strains were found to be severely defective in DmpR‐mediated transcription, demonstrating the requirement for this metabolic signal. A subset of mutations in the β, β′ and σ70 subunits of RNA polymerase, which confer prototrophy on ppGpp0E. coli, was also found to restore specific DmpR‐mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the σ54‐RNA polymerase. These data provide a direct mechanistic link between the physiological status of the cell and expression from σ54 promoters.

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.

(p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB.

In this report we have examined the role of the regulatory alarmone (p)ppGpp on expression of virulence determinants of uropathogenic Escherichia coli strains. The ability to form biofilms is shown to be markedly diminished in (p)ppGpp‐deficient strains. We present evidence (i) that (p)ppGpp tightly regulates expression of the type 1 fimbriae in both commensal and pathogenic E. coli isolates by increasing the subpopulation of cells that express the type 1 fimbriae; and (ii) that the effect of (p)ppGpp on the number of fimbrial expressing cells can ultimately be traced to its role in transcription of the fimB recombinase gene, whose product mediates inversion of the fim promoter to the productive (ON) orientation. Primer extension analysis suggests that the effect of (p)ppGpp on transcription of fimB occurs by altering the activity of only one of the two fimB promoters. Furthermore, spontaneous mutants with properties characteristic of ppGpp0 suppressors restore fimB transcription and consequent downstream effects in the absence of (p)ppGpp. Consistently, the rpoB3770 allele also fully restores transcription of fimB in a ppGpp0 strain and artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation. Our findings suggest that the (p)ppGpp‐stimulated expression of type 1 fimbriae may be relevant during the interaction of pathogenic E. coli with the host.

Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo

The expression of type 1 fimbriae is dependent on the intracellular levels of ppGpp through stimulation of fimB transcription. Here we show that in contrast to the previously described decreased fimbriation observed in a ppGpp‐deficient strain, DksA deficiency results in a hyperfimbriated state. In vivo assays show that the effect of DksA deficiency on the type 1 fimbriae occurs at the phase variation level because of elevated transcription from the fimB P2 promoter. In contrast, our in vitro transcription studies demonstrate that ppGpp and DksA can stimulate transcription from the fimB P2 promoter both independently and codependently. We provide evidences that the apparently contradictory results from the in vivo and in vitro transcriptional studies are at least in part a consequence of the increased association of the anti‐pausing factors (GreA and GreB) to the RNA polymerase in the absence of DksA in vivo.

Nucleotide sequences of the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas CF600

The nucleotide sequence of a 2493 base pair (bp) region, spanning the coding regions for the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), was determined. The deduced protein sequence for HMSD is 486 amino acid residues long with an Mr of 51,682. HMSD has homology with a number of aldehyde dehydrogenases from various eukaryotic sources. The deduced protein sequence for HMSH is 283 amino acids long with an Mr of 30,965. The amino acid composition of this enzyme is similar to that of isofunctional enzymes from toluene and m-cresol catabolic pathways.

Nucleotide sequence and expression of the catechol 2,3-dioxygenase-encoding gene of phenol-catabolizing Pseudomonas CF600.

Pseudomonas CF600 degrades phenol and some of its methylated derivatives via a plasmid-encoded catabolic pathway. The catechol 2,3-dioxygenase (C23O) enzyme of this pathway catalyses the conversion of catechol to 2-hydroxymuconic semialdehyde. We have determined the nucleotide (nt) sequence of the dmpB structural gene for this enzyme, and expressed and identified its polypeptide product in Escherichia coli. The xylE gene of TOL plasmid pWWO and the nahH gene of plasmid NAH7 encode analogous C23O enzymes. Comparison of these three genes shows homology of 78-81% on the nt level and 83-87% homology on the amino acid level.