SignificanceTranscription in Gram-negative bacteria is greatly influenced by the synergistic interactions of DksA and the nucleotide alarmone guanosine tetraphosphate. Our investigations reveal a unique, previously unknown layer of transcriptional regulation that depends on redox-based protein–protein interactions between DksA and the molecular chaperone DnaJ. Tripartite connections between DksA, DnaJ, and guanosine tetraphosphate afford a dynamic range of transcriptional responses to H2O2 concentrations associated with redox signaling or oxidative stress. The redox-based gene regulation dependent on the combined actions of DksA, DnaJ, and guanosine tetraphosphate confers resistance of Salmonella to the antimicrobial activity of the NADPH phagocyte oxidase.
Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa's response to the host, particularly changes regulated by the hostderived quaternary amines choline and glycine betaine (GB). We previously identified GbdR as an AraC family transcription factor that directly regulates choline acquisition from host phospholipids (via binding to plcH and pchP promoters), is required for catabolism of the choline metabolite GB, and is an activator that induces transcription in response to GB or dimethylglycine. Our goal was to characterize the GbdR regulon in P. aeruginosa by using genetics and chemical biology in combination with transcriptomics and in vitro DNA-binding assays. Here we show that GbdR activation regulates transcription of 26 genes from 12 promoters, 11 of which have measureable binding to GbdR in vitro. The GbdR regulon includes the genes encoding GB, dimethylglycine, sarcosine, glycine, and serine catabolic enzymes and the BetX and CbcXWV quaternary amine transport proteins. We characterized the GbdR consensus binding site and used it to identify that the recently characterized acetylcholine esterase gene, choE (PA4921), is also regulated by GbdR. The regulon member not directly controlled by GbdR is the secreted lipase gene lipA, which was also the only regulon member repressed under GbdR-activating conditions. Determination of the GbdR regulon provides deeper understanding of how GbdR links bacterial metabolism and virulence. Additionally, identification of two uncharacterized regulon members suggests roles for these proteins in response to choline metabolites.
The adaptations that protect pathogenic microorganisms against the cytotoxicity of nitric oxide (NO) engendered in the immune response are incompletely understood. We show here that salmonellae experiencing nitrosative stress suffer dramatic losses of the nucleoside triphosphates ATP, GTP, CTP, and UTP while simultaneously generating a massive burst of the alarmone nucleotide guanosine tetraphosphate. RelA proteins associated with ribosomes overwhelmingly synthesize guanosine tetraphosphate in response to NO as a feedback mechanism to transient branched-chain amino acid auxotrophies. Guanosine tetraphosphate activates the transcription of valine biosynthetic genes, thereby reestablishing branched-chain amino acid biosynthesis that enables the translation of the NO-consuming flavohemoglobin Hmp. Guanosine tetraphosphate synthesized by RelA protects salmonellae from the metabolic stress inflicted by reactive nitrogen species generated in the mammalian host response. This research illustrates the importance of nucleotide metabolism in the adaptation of salmonellae to the nutritional stress imposed by NO released in the innate host response. Nitric oxide triggers dramatic drops in nucleoside triphosphates, the building blocks that power DNA replication; RNA transcription; translation; cell division; and the biosynthesis of fatty acids, lipopolysaccharide, and peptidoglycan. Concomitantly, this diatomic gas stimulates a burst of guanosine tetraphosphate. Global changes in nucleotide metabolism may contribute to the potent bacteriostatic activity of nitric oxide. In addition to inhibiting numerous growth-dependent processes, guanosine tetraphosphate positively regulates the transcription of branched-chain amino acid biosynthesis genes, thereby facilitating the translation of antinitrosative defenses that mediate recovery from nitrosative stress.
bAlgR is a key Pseudomonas aeruginosa transcriptional response regulator required for virulence. AlgR activates alginate production and twitching motility but represses the Rhl quorum-sensing (QS) system, including rhamnolipid production. The role of AlgR phosphorylation is enigmatic, since phosphorylated AlgR (AlgR-P) is required for twitching motility through the fimU promoter but is not required for the activation of alginate production. In order to examine the role of AlgR phosphorylation in vivo, a PAO1 algRD54E strain (with algR encoding a D-to-E change at position 54), which constitutively activates fimU transcription and exhibits twitching motility, was created. A corresponding PAO1 algRD54N strain (with algR encoding a D-to-N change at position 54) that does not activate fimU or twitching motility was compared to PAO1, PAO1 algRD54E, PAO1 ⌬algZ (deletion of the algZ [fimS] gene, encoding a putative histidine kinase), and PAO1 ⌬algR for swarming motility, rhamnolipid production, and rhlA transcription. PAO1 and PAO1 algRD54E produced approximately 2-fold-higher levels of rhamnolipids than PAO1 algRD54N and PAO1 ⌬algZ, thereby indicating that phosphorylated AlgR is required for normal rhamnolipid production. Examination of purified AlgR, AlgR-P, AlgR D54N, and AlgR D54E showed that AlgR-P and AlgR D54E bound preferentially to the fimU and rhlA promoters. Additionally, AlgR-P bound specifically to two sites within the rhlA promoter that were not bound by unphosphorylated AlgR. Taken together, these results indicate that phosphorylated AlgR-P has increased affinity for the rhlA promoter and is required for the coordinate activation of twitching motility, rhamnolipid production, and swarming motility in P. aeruginosa.
The microbial adaptations to the respiratory burst remain poorly understood, and establishing how the NADPH oxidase (NOX2) kills microbes has proven elusive. Here we demonstrate that NOX2 collapses the ΔpH of intracellular Salmonella Typhimurium. The depolarization experienced by Salmonella undergoing oxidative stress impairs folding of periplasmic proteins. Depolarization in respiring Salmonella mediates intense bactericidal activity of reactive oxygen species (ROS). Salmonella adapts to the challenges oxidative stress imposes on membrane bioenergetics by shifting redox balance to glycolysis and fermentation, thereby diminishing electron flow through the membrane, meeting energetic requirements and anaplerotically generating tricarboxylic acid intermediates. By diverting electrons away from the respiratory chain, glycolysis also enables thiol/disulfide exchange-mediated folding of bacterial cell envelope proteins during periods of oxidative stress. Thus, primordial metabolic pathways, already present in bacteria before aerobic respiration evolved, offer a solution to the stress ROS exert on molecular targets at the bacterial cell envelope.
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