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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., (Copyright © 2020 Irie, La Mensa, Murina, Hauryliuk, Tenson and Shingler.)

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 σ 54 -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 σ 54 -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., (© 2020 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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 σ 54 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 σ 54 -dependent transcription.

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 σ 54 -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., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Bacteria swim and swarm using rotating flagella that are driven by a membrane-spanning motor complex. Performance of the flagella motility apparatus is modulated by the chemosensory signal transduction system to allow navigation through physico-chemical gradients - a process that can be fine-tuned by the bacterial second messenger c-di-GMP. We have previously analysed the Pseudomonas putida signalling protein PP2258 that has the capacity to both synthesize and degrade c-di-GMP. A PP2258 null mutant displays reduced motility, implicating the c-di-GMP signal originating from this protein in control of P. putida motility. In Escherichia coli and Salmonella, the PilZ-domain protein YcgR mediates c-di-GMP responsive control of motility through interaction with the flagellar motors. Here we provide genetic evidence that the P. putida protein PP4397 (also known as FlgZ), despite low sequence homology and a different genomic context to YcgR, functions as a c-di-GMP responsive link between the signal arising from PP2258 and alterations in swimming and swarming motility in P. putida.

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., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Biofilm dispersal is a genetically programmed response enabling bacterial cells to exit the biofilm in response to particular physiological or environmental conditions. In Pseudomonas putida biofilms, nutrient starvation triggers c-di-GMP hydrolysis by phosphodiesterase BifA, releasing inhibition of protease LapG by the c-di-GMP effector protein LapD, and resulting in proteolysis of the adhesin LapA and the subsequent release of biofilm cells. Here we demonstrate that the stringent response, a ubiquitous bacterial stress response, is accountable for relaying the nutrient stress signal to the biofilm dispersal machinery. Mutants lacking elements of the stringent response - (p)ppGpp sythetases [RelA and SpoT] and/or DksA - were defective in biofilm dispersal. Ectopic (p)ppGpp synthesis restored biofilm dispersal in a ∆relA ∆spoT mutant. In vivo gene expression analysis showed that (p)ppGpp positively regulates transcription of bifA, and negatively regulates transcription of lapA and the lapBC, and lapE operons, encoding a LapA-specific secretion system. Further in vivo and in vitro characterization revealed that the PbifA promoter is dependent on the flagellar σ factor FliA, and positively regulated by ppGpp and DksA. Our results indicate that the stringent response stimulates biofilm dispersal under nutrient limitation by coordinately promoting LapA proteolysis and preventing de novo LapA synthesis and secretion.

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 alarmone nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance and virulence, making (p)ppGpp-mediated signaling a promising target for development of antibacterials. Although ppGpp itself is an activator of the ribosome-associated ppGpp synthetase RelA, several ppGpp mimics have been developed as RelA inhibitors. However promising, the currently available ppGpp mimics are relatively inefficient, with IC 50 in the sub-mM range. In an attempt to identify a potent and specific inhibitor of RelA capable of abrogating (p)ppGpp production in live bacterial cells, we have tested a targeted nucleotide library using a biochemical test system comprised of purified Escherichia coli components. While none of the compounds fulfilled this aim, the screen has yielded several potentially useful molecular tools for biochemical and structural work., Competing Interests: The authors declare no competing financial interests.

Divergent transcription from within bacterial intergenic regions frequently involves promoters dependent on alternative σ-factors. This is the case for the non-overlapping σ 70 - and σ 54 -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 σ 54 -promoter as the mechanism underlying inter-promoter communication that results in stimulation of the activity of the σ 70 -promoter. The non-overlapping 'back-to-back' configuration of a powerful σ 54 -promoter and weak σ 70 -promoter within this system offers a previously unknown means of inter-sigmulon communication that renders the σ 70 -promoter subservient to signals that elicit σ 54 -dependent transcription without it possessing a cognate binding site for the σ 54 -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., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)