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.

Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), together named (p)ppGpp, regulate diverse aspects of Salmonella pathogenesis, including synthesis of nutrients, resistance to inflammatory mediators, and expression of secretion systems. In Salmonella, these nucleotide alarmones are generated by the synthetase activities of RelA and SpoT proteins. In addition, the (p)ppGpp hydrolase activity of the bifunctional SpoT protein is essential to preserve cell viability. The contribution of SpoT to physiology and pathogenesis has proven elusive in organisms such as Salmonella, because the hydrolytic activity of this RelA and SpoT homologue (RSH) is vital to prevent inhibitory effects of (p)ppGpp produced by a functional RelA. Here, we describe the biochemical and functional characterization of a spoT-Δctd mutant Salmonella strain encoding a SpoT protein that lacks the C-terminal regulatory elements collectively referred to as “ctd.” Salmonella expressing the spoT-Δctd variant hydrolyzes (p)ppGpp with similar kinetics to those of wild-type bacteria, but it is defective at synthesizing (p)ppGpp in response to acidic pH. Salmonella spoT-Δctd mutants have virtually normal adaptations to nutritional, nitrosative, and oxidative stresses, but poorly induce metal cation uptake systems and Salmonella pathogenicity island 2 (SPI-2) genes in response to the acidic pH of the phagosome. Importantly, spoT-Δctd mutant Salmonella replicates poorly intracellularly and is attenuated in a murine model of acute salmonellosis. Collectively, these investigations indicate that (p)ppGpp synthesized by SpoT serves a unique function in the adaptation of Salmonella to the intracellular environment of host phagocytes that cannot be compensated by the presence of a functional RelA. IMPORTANCE Pathogenic bacteria experience nutritional challenges during colonization and infection of mammalian hosts. Binding of the alarmone nucleotide guanosine tetraphosphate (ppGpp) to RNA polymerase coordinates metabolic adaptations and virulence gene transcription, increasing the fitness of diverse Gram-positive and Gram-negative bacteria as well as that of actinomycetes. Gammaproteobacteria such as Salmonella synthesize ppGpp by the combined activities of the closely related RelA and SpoT synthetases. Due to its profound inhibitory effects on growth, ppGpp must be removed; in Salmonella, this process is catalyzed by the vital hydrolytic activity of the bifunctional SpoT protein. Because SpoT hydrolase activity is essential in cells expressing a functional RelA, we have a very limited understanding of unique roles these two synthetases may assume during interactions of bacterial pathogens with their hosts. We describe here a SpoT truncation mutant that lacks ppGpp synthetase activity and all C-terminal regulatory domains but retains excellent hydrolase activity. Our studies of this mutant reveal that SpoT uniquely senses the acidification of phagosomes, inducing virulence programs that increase Salmonella fitness in an acute model of infection. Our investigations indicate that the coexistence of RelA/SpoT homologues in a bacterial cell is driven by the need to mount a stringent response to a myriad of physiological and host-specific signatures.

Detoxification, scavenging, and repair systems embody the archetypical antioxidant defenses of prokaryotic and eukaryotic cells. Metabolic rewiring also aids with the adaptation of bacteria to oxidative stress. Evolutionarily diverse bacteria combat the toxicity of reactive oxygen species (ROS) by actively engaging the stringent response, a stress program that controls many metabolic pathways at the level of transcription initiation via guanosine tetraphosphate and the α-helical DksA protein. Studies herein with Salmonella demonstrate that the interactions of structurally related, but functionally unique, α-helical Gre factors with the secondary channel of RNA polymerase elicit the expression of metabolic signatures that are associated with resistance to oxidative killing. Gre proteins both improve transcriptional fidelity of metabolic genes and resolve pauses in ternary elongation complexes of Embden–Meyerhof–Parnas (EMP) glycolysis and aerobic respiration genes. The Gre-directed utilization of glucose in overflow and aerobic metabolism satisfies the energetic and redox demands of Salmonella, while preventing the occurrence of amino acid bradytrophies. The resolution of transcriptional pauses in EMP glycolysis and aerobic respiration genes by Gre factors safeguards Salmonella from the cytotoxicity of phagocyte NADPH oxidase in the innate host response. In particular, the activation of cytochrome bd protects Salmonella from phagocyte NADPH oxidase-dependent killing by promoting glucose utilization, redox balancing, and energy production. Control of transcription fidelity and elongation by Gre factors represent important points in the regulation of metabolic programs supporting bacterial pathogenesis.

The exquisite specificity between a sensor kinase and its cognate response regulator ensures faithful partner selectivity within two-component pairs concurrently firing in a single bacterium, minimizing crosstalk with other members of this conserved family of paralogous proteins. We show that conserved hydrophobic and charged residues on the surface of thioredoxin serve as a docking station for structurally diverse response regulators. Using the OmpR protein, we identify residues in the flexible linker and the C-terminal β-hairpin that enable associations of this archetypical response regulator with thioredoxin, but are dispensable for interactions of this transcription factor to its cognate sensor kinase EnvZ, DNA or RNA polymerase. Here we show that the promiscuous interactions of response regulators with thioredoxin foster the flow of information through otherwise highly dedicated two-component signaling systems, thereby enabling both the transcription of Salmonella pathogenicity island-2 genes as well as growth of this intracellular bacterium in macrophages and mice.

Salmonella invades host cells and replicates inside acidified, remodeled vacuoles that are exposed to reactive oxygen species (ROS) generated by the innate immune response. Oxidative products of the phagocyte NADPH oxidase mediate antimicrobial activity, in part, by collapsing the ΔpH of intracellular Salmonella .