Elevated levels of VCA0117 (VasH) in response to external signals activate the type VI secretion system of Vibrio cholerae O1 El Tor A1552.

The type VI nanomachine is critical for Vibrio cholerae to establish infections and to thrive in niches co-occupied by competing bacteria. The genes for the type VI structural proteins are encoded in one large and two small auxiliary gene clusters. VCA0117 (VasH) - a σ54 -transcriptional activator - is strictly required for functionality of the type VI secretion system since it controls production of the structural protein Hcp. While some strains constitutively produce a functional system, others do not and require specific growth conditions of low temperature and high osmolarity for expression of the type VI machinery. Here, we trace integration of these regulatory signals to the promoter activity of the large gene cluster in which many components of the machinery and VCA0117 itself are encoded. Using in vivo and in vitro assays and variants of VCA0117, we show that activation of the σ54 -promoters of the auxiliary gene clusters by elevated VCA0117 levels are all that is required to overcome the need for specialized growth conditions. We propose a model in which signal integration via the large operon promoter directs otherwise restrictive levels of VCA0117 that ultimately dictates a sufficient supply of Hcp for completion of a functional type VI secretion system.

Negative allosteric regulation of Enterococcus faecalis small alarmone synthetase RelQ by single-stranded RNA.

The alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp, are key regulators of bacterial growth, stress adaptation, pathogenicity, and antibiotic tolerance. We show that the tetrameric small alarmone synthetase (SAS) RelQ from the Gram-positive pathogen Enterococcus faecalis is a sequence-specific RNA-binding protein. RelQ's enzymatic and RNA binding activities are subject to intricate allosteric regulation. (p)ppGpp synthesis is potently inhibited by the binding of single-stranded RNA. Conversely, RelQ's enzymatic activity destabilizes the RelQ:RNA complex. pppGpp, an allosteric activator of the enzyme, counteracts the effect of RNA. Tetramerization of RelQ is essential for this regulatory mechanism, because both RNA binding and enzymatic activity are abolished by deletion of the SAS-specific C-terminal helix 5α. The interplay of pppGpp binding, (p)ppGpp synthesis, and RNA binding unites two archetypal regulatory paradigms within a single protein. The mechanism is likely a prevalent but previously unappreciated regulatory switch used by the widely distributed bacterial SAS enzymes.

The Y233 gatekeeper of DmpR modulates effector-responsive transcriptional control of σ54 -RNA polymerase.

DmpR is the obligate transcriptional activator of genes involved in (methyl)phenol catabolism by Pseudomonas putida. DmpR belongs to the AAA+ class of mechano-transcriptional regulators that employ ATP-hydrolysis to engage and remodel σ54 -RNA polymerase to allow transcriptional initiation. Previous work has established that binding of phenolic effectors by DmpR is a prerequisite to relieve interdomain repression and allow ATP-binding to trigger transition to its active multimeric conformation, and further that a structured interdomain linker between the effector- and ATP-binding domains is involved in coupling these processes. Here, we present evidence from ATPase and in vivo and in vitro transcription assays that a tyrosine residue of the interdomain linker (Y233) serves as a gatekeeper to constrain ATP-hydrolysis and aromatic effector-responsive transcriptional activation by DmpR. An alanine substitution of Y233A results in both increased ATPase activity and enhanced sensitivity to aromatic effectors. We propose a model in which effector-binding relocates Y233 to synchronize signal-reception with multimerisation to provide physiologically 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 and has implications for development of enhanced sensitivity of biosensor used to detect pollutants.

Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600.

The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here, we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.

Inter-sigmulon communication through topological promoter coupling.

Divergent transcription from within bacterial intergenic regions frequently involves promoters dependent on alternative σ-factors. This is the case for the non-overlapping σ70- and σ54-dependent promoters that control production of the substrate-responsive regulator and enzymes for (methyl)phenol catabolism. Here, using an array of in vivo and in vitro assays, we identify transcription-driven supercoiling arising from the σ54-promoter as the mechanism underlying inter-promoter communication that results in stimulation of the activity of the σ70-promoter. The non-overlapping 'back-to-back' configuration of a powerful σ54-promoter and weak σ70-promoter within this system offers a previously unknown means of inter-sigmulon communication that renders the σ70-promoter subservient to signals that elicit σ54-dependent transcription without it possessing a cognate binding site for the σ54-RNA polymerase holoenzyme. This mode of control has the potential to be a prevalent, but hitherto unappreciated, mechanism by which bacteria adjust promoter activity to gain appropriate transcriptional control.

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.

AccR is a master regulator involved in carbon catabolite repression of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB.

Here we characterized the first known transcriptional regulator that accounts for carbon catabolite repression (CCR) control of the anaerobic catabolism of aromatic compounds in bacteria. The AccR response regulator of Azoarcus sp. CIB controls succinate-responsive CCR of the central pathways for the anaerobic catabolism of aromatics by this strain. Phosphorylation of AccR to AccR-P triggers a monomer-to-dimer transition as well as the ability to bind to the target promoter and causes repression both in vivo and in vitro. Substitution of the Asp(60) phosphorylation target residue of the N-terminal receiver motif of AccR to a phosphomimic Glu residue generates a constitutively active derivative that behaves as a superrepressor of the target genes. AccR-P binds in vitro to a conserved inverted repeat (ATGCA-N6-TGCAT) present at two different locations within the PN promoter of the bzd genes for anaerobic benzoate degradation. Because the DNA binding-proficient C-terminal domain of AccR is monomeric, we propose an activation mechanism in which phosphorylation of Asp(60) of AccR alleviates interdomain repression mediated by the N-terminal domain. The presence of AccR-like proteins encoded in the genomes of other ß-proteobacteria of the Azoarcus/Thauera group further suggests that AccR constitutes a master regulator that controls anaerobic CCR in these bacteria.

Transcriptional and translational control through the 5'-leader region of the dmpR master regulatory gene of phenol metabolism.

Expression of pathways for dissimilation of toxic aromatic compounds such as (methyl)phenols interfaces both stress-response and carbon catabolite repression control cascades. In Pseudomonas putida, carbon catabolite repression is mediated by the protein Crc - a translational repressor that counteracts utilization of less-preferred carbon sources as growth substrates until they are needed. In this work we dissect the regulatory role of the 5'-leader region (5'-LR) of the dmpR gene that encodes the master regulator of (methyl)phenol catabolism. Using deletion and substitution mutants combined with artificial manipulations of Crc availability in P. putida, we present evidence that a DNA motif within the 5'-leader region is critical for inhibition of the output from the Pr promoter that drives transcription of dmpR, while the RNA chaperone Hfq facilitates Crc-mediated translation repression through the 5'-leader region of the dmpR mRNA. The results are discussed in the light of a model in which Hfq assists Crc to target a sequence within a loop formed by secondary structure of the 5'-LR mRNA. Our results support the idea that Crc functions as a global translational inhibitor to co-ordinate hierarchical carbon utilization in Pseudomonads.

The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs.

The Crc protein is a global regulator that has a key role in catabolite repression and optimization of metabolism in Pseudomonads. Crc inhibits gene expression post-transcriptionally, preventing translation of mRNAs bearing an AAnAAnAA motif [the catabolite activity (CA) motif] close to the translation start site. Although Crc was initially believed to bind RNA by itself, this idea was recently challenged by results suggesting that a protein co-purifying with Crc, presumably the Hfq protein, could account for the detected RNA-binding activity. Hfq is an abundant protein that has a central role in post-transcriptional gene regulation. Herein, we show that the Pseudomonas putida Hfq protein can recognize the CA motifs of RNAs through its distal face and that Crc facilitates formation of a more stable complex at these targets. Crc was unable to bind RNA in the absence of Hfq. However, pull-down assays showed that Crc and Hfq can form a co-complex with RNA containing a CA motif in vitro. Inactivation of the hfq or the crc gene impaired catabolite repression to a similar extent. We propose that Crc and Hfq cooperate in catabolite repression, probably through forming a stable co-complex with RNAs containing CA motifs to result in inhibition of translation initiation.

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.

An A-tract at the AtzR binding site assists DNA binding, inducer-dependent repositioning and transcriptional activation of the PatzDEF promoter.

The LysR-type regulator AtzR activates the Pseudomonas sp. ADP atzDEF operon in response to nitrogen limitation and cyanuric acid. Activation involves repositioning of the AtzR tetramer on the PatzDEF promoter and relaxation of an AtzR-induced DNA bend. Here we examine the in vivo and in vitro contribution of an A5 -tract present at the PatzDEF promoter region to AtzR binding and transcriptional activation. Substitution of the A-tract for the sequence ACTCA prevented PatzDEF activation and high-affinity AtzR binding, impaired AtzR contacts with the activator binding site and shifted the position of the AtzR-induced DNA bend. Analysis of a collection of mutants bearing different alterations in the A-tract sequence showed that the extent of AtzR-dependent activation does not correlate with the magnitude or orientation of the spontaneous DNA bend generated at this site. Our results support the notion that indirect readout of the A-tract-associated narrow minor groove is essential for the AtzR-DNA complex to achieve a conformation competent for activation of the PatzDEF promoter. Conservation of this motif in several binding sites of LysR-type regulators suggests that this mechanism may be shared by other proteins in this family.

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 σ(70)-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 σ(70)-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.

A hyper-mutant of the unusual sigma70-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 σ(70)-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 σ(70)-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.

Activation and repression of a sigmaN-dependent promoter naturally lacking upstream activation sequences.

The Pseudomonas sp. strain ADP protein AtzR is a LysR-type transcriptional regulator required for activation of the atzDEF operon in response to nitrogen limitation and cyanuric acid. Transcription of atzR is directed by the sigma(N)-dependent promoter PatzR, activated by NtrC and repressed by AtzR. Here we use in vivo and in vitro approaches to address the mechanisms of PatzR activation and repression. Activation by NtrC did not require any promoter sequences other than the sigma(N) recognition motif both in vivo and in vitro, suggesting that NtrC activates PatzR in an upstream activation sequences-independent fashion. Regarding AtzR-dependent autorepression, our in vitro transcription experiments show that the concentration of AtzR required for repression of the PatzR promoter in vitro correlates with AtzR affinity for its binding site. In addition, AtzR prevents transcription from PatzR when added to a preformed E-sigma(N)-PatzR closed complex, but isomerization to an open complex prevents repression. Gel mobility shift and DNase I footprint assays indicate that DNA-bound AtzR and E-sigma(N) are mutually exclusive. Taken together, these results strongly support the notion that AtzR represses transcription from PatzR by competing with E-sigma(N) for their overlapping binding sites. There are no previous reports of a similar mechanism for repression of sigma(N)-dependent transcription.

The sigma-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.

Hfq-Assisted RsmA Regulation Is Central to Pseudomonas aeruginosa Biofilm Polysaccharide PEL Expression.

To appropriately switch between sessile and motile lifestyles, bacteria control expression of biofilm-associated genes through multiple regulatory elements. In Pseudomonas aeruginosa, the post-transcriptional regulator RsmA has been implicated in the control of various genes including those related to biofilms, but much of the evidence for these links is limited to transcriptomic and phenotypic studies. RsmA binds to target mRNAs to modulate translation by affecting ribosomal access and/or mRNA stability. Here, we trace a global regulatory role of RsmA to inhibition of the expression of Vfr-a transcription factor that inhibits transcriptional regulator FleQ. FleQ directly controls biofilm-associated genes that encode the PEL polysaccharide biosynthesis machinery. Furthermore, we show that RsmA alone cannot bind vfr mRNA but requires the assistance of RNA chaperone protein Hfq. This is the first example where a RsmA protein family member requires another protein for binding to its target RNA.

Tetrameric architecture of an active phenol-bound form of the AAA+ transcriptional regulator DmpR.

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.

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.

The styrene-responsive StyS/StyR regulation system controls expression of an auxiliary phenylacetyl-coenzyme A ligase: implications for rapid metabolic coupling of the styrene upper- and lower-degradative pathways.

Pseudomonas sp. strain Y2 degrades styrene through oxidation to phenylacetic acid via the styABCD operon-encoded enzymes, whose expression is induced in response to styrene by the StyS/StyR two-component regulatory system. Further transformation of phenylacetic acid to tricarboxylic acid cycle intermediates is mediated by the enzymes of paa catabolic genes, whose expression is regulated by the PaaX repressor. The first step of this paa degradation pathway is catalysed by paaF-encoded phenylacetyl-coenzyme A ligases that produce phenylacetyl-coenzyme A. This metabolic intermediate, upon being bound by PaaX, inactivates PaaX-mediated repression of both the paa genes and the styABCD operon. Strain Y2 is unique in having three paaF genes located within two complete copies of the paa gene clusters. Expression of both paaF and paaF3 is controlled by the PaaX repressor. Here we use specific mutants in combination with in vivo and in vitro assays to demonstrate that paaF2, adjacent to the StyS/StyR regulatory genes, belongs to the StyR regulon and is not subject to repression by PaaX. We propose that this unexpected styrene-responsive regulatory strategy for the otherwise metabolically redundant PaaF2 auxiliary enzyme provides a system for rapid co-ordinate de-repression of the two sets of catabolic genes required for styrene degradation.

sigma54-RNA polymerase controls sigma70-dependent transcription from a non-overlapping divergent promoter.

Divergent transcription of a regulatory gene and a cognate promoter under its control is a common theme in bacterial regulatory circuits. This genetic organization is found for the dmpR gene that encodes the substrate-responsive specific regulator of the sigma(54)-dependent Po promoter, which controls (methyl)phenol catabolism. Here we identify the Pr promoter of dmpR as a sigma(70)-dependent promoter that is regulated by a novel mechanism in which sigma(54)-RNA polymerase occupancy of the non-overlapping sigma(54)-Po promoter stimulates sigma(70)-Pr output. In addition, we show that DmpR stimulates its own production through Po activity both in vivo and in vitro. Hence, the demonstrated regulatory circuit reveals a novel role for sigma(54)-RNA polymerase, namely regulation of a sigma(70)-dependent promoter, and a new mechanism that places a single promoter under dual control of two alternative forms of RNA polymerase. We present a model in which guanosine tetra-phosphate plays a major role in the interplay between sigma(54)- and sigma(70)-dependent transcription to ensure metabolic integration to couple sigma(70)-Pr output to both low-energy conditions and the presence of substrate.