A bacterial virulence protein promotes pathogenicity by inhibiting the bacterium's own F1Fo ATP synthase.

Several intracellular pathogens, including Salmonella enterica and Mycobacterium tuberculosis, require the virulence protein MgtC to survive within macrophages and to cause a lethal infection in mice. We now report that, unlike secreted virulence factors that target the host vacuolar ATPase to withstand phagosomal acidity, the MgtC protein acts on Salmonella's own F1Fo ATP synthase. This complex couples proton translocation to ATP synthesis/hydrolysis and is required for virulence. We establish that MgtC interacts with the a subunit of the F1Fo ATP synthase, hindering ATP-driven proton translocation and NADH-driven ATP synthesis in inverted vesicles. An mgtC null mutant displays heightened ATP levels and an acidic cytoplasm, whereas mgtC overexpression decreases ATP levels. A single amino acid substitution in MgtC that prevents binding to the F1Fo ATP synthase abolishes control of ATP levels and attenuates pathogenicity. MgtC provides a singular example of a virulence protein that promotes pathogenicity by interfering with another virulence protein.

Gut colonization by Bacteroides requires translation by an EF-G paralog lacking GTPase activity.

Protein synthesis is crucial for cell growth and survival yet one of the most energy-consuming cellular processes. How, then, do cells sustain protein synthesis under starvation conditions when energy is limited? To accelerate the translocation of mRNA-tRNAs through the ribosome, bacterial elongation factor G (EF-G) hydrolyzes energy-rich guanosine triphosphate (GTP) for every amino acid incorporated into a protein. Here, we identify an EF-G paralog-EF-G2-that supports translocation without hydrolyzing GTP in the gut commensal bacterium Bacteroides thetaiotaomicron. EF-G2's singular ability to sustain protein synthesis, albeit at slow rates, is crucial for bacterial gut colonization. EF-G2 is ~10-fold more abundant than canonical EF-G1 in bacteria harvested from murine ceca and, unlike EF-G1, specifically accumulates during carbon starvation. Moreover, we uncover a 26-residue region unique to EF-G2 that is essential for protein synthesis, EF-G2 dissociation from the ribosome, and responsible for the absence of GTPase activity. Our findings reveal how cells curb energy consumption while maintaining protein synthesis to advance fitness in nutrient-fluctuating environments.

Chaperone Hsp70 helps Salmonella survive infection-relevant stress by reducing protein synthesis.

In all domains of life, Hsp70 chaperones preserve protein homeostasis by promoting protein folding and degradation and preventing protein aggregation. We now report that the Hsp70 from the bacterial pathogen Salmonella enterica serovar Typhimurium-termed DnaK-independently reduces protein synthesis in vitro and in S. Typhimurium facing cytoplasmic Mg2+ starvation, a condition encountered during infection. This reduction reflects a 3-fold increase in ribosome association with DnaK and a 30-fold decrease in ribosome association with trigger factor, the chaperone normally associated with translating ribosomes. Surprisingly, this reduction does not involve J-domain cochaperones, unlike previously known functions of DnaK. Removing the 74 C-terminal amino acids of the 638-residue long DnaK impeded DnaK association with ribosomes and reduction of protein synthesis, rendering S. Typhimurium defective in protein homeostasis during cytoplasmic Mg2+ starvation. DnaK-dependent reduction in protein synthesis is critical for survival against Mg2+ starvation because inhibiting protein synthesis in a dnaK-independent manner overcame the 10,000-fold loss in viability resulting from DnaK truncation. Our results indicate that DnaK protects bacteria from infection-relevant stresses by coordinating protein synthesis with protein folding capacity.

Activator of one protease transforms into inhibitor of another in response to nutritional signals.

All cells use proteases to adjust protein amounts. Proteases maintain protein homeostasis by degrading nonfunctional toxic proteins and play regulatory roles by targeting particular substrates in response to specific signals. Here we address how cells tune protease specificity to nutritional signals. We report that Salmonella enterica increases the specificity of the broadly conserved proteases Lon and ClpSAP by transforming the Lon activator and substrate HspQ into an inhibitor of the N-degron recognin ClpS, the adaptor of the ClpAP protease. We establish that upon acetylation, HspQ stops being a Lon activator and substrate and that the accumulated HspQ binds to ClpS, hindering degradation of ClpSAP substrates. Growth on glucose promotes HspQ acetylation by increasing acetyl-CoA amounts, thereby linking metabolism to proteolysis. By altering protease specificities but continuing to degrade junk proteins, cells modify the abundance of particular proteins while preserving the quality of their proteomes. This rapid response mechanism linking protease specificity to nutritional signals is broadly conserved.

Cellular Adaptations to Cytoplasmic Mg2+ Limitation.

Mg2+ is the most abundant divalent cation in living cells. It is essential for charge neutralization, macromolecule stabilization, and the assembly and activity of ribosomes and as a cofactor for enzymatic reactions. When experiencing low cytoplasmic Mg2+, bacteria adopt two main strategies: They increase the abundance and activity of Mg2+ importers and decrease the abundance of Mg2+-chelating ATP and rRNA. These changes reduce regulated proteolysis by ATP-dependent proteases and protein synthesis in a systemic fashion. In many bacterial species, the transcriptional regulator PhoP controls expression of proteins mediating these changes. The 5' leader region of some mRNAs responds to low cytoplasmic Mg2+ or to disruptions in translation of open reading frames in the leader regions by furthering expression of the associated coding regions, which specify proteins mediating survival when the cytoplasmic Mg2+ concentration is low. Microbial species often utilize similar adaptation strategies to cope with low cytoplasmic Mg2+ despite relying on different genes to do so.

Protein synthesis controls phosphate homeostasis.

Phosphorus is an essential element assimilated largely as orthophosphate (Pi). Cells respond to Pi starvation by importing Pi from their surroundings. We now report that impaired protein synthesis alone triggers a Pi starvation response even when Pi is plentiful in the extracellular milieu. In the bacterium Salmonella enterica serovar Typhimurium, this response entails phosphorylation of the regulatory protein PhoB and transcription of PhoB-dependent Pi transporter genes and is eliminated upon stimulation of adenosine triphosphate (ATP) hydrolysis. When protein synthesis is impaired due to low cytoplasmic magnesium (Mg2+), Salmonella triggers the Pi starvation response because ribosomes are destabilized, which reduces ATP consumption and thus free cytoplasmic Pi. This response is transient because low cytoplasmic Mg2+ promotes an uptake in Mg2+ and a decrease in ATP levels, which stabilizes ribosomes, resulting in ATP consumption and Pi increase, thus ending the response. Notably, pharmacological inhibition of protein synthesis also elicited a Pi starvation response in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae Our findings identify a regulatory connection between protein synthesis and Pi homeostasis that is widespread in nature.

A bacterial mRNA leader that employs different mechanisms to sense disparate intracellular signals.

Bacterial mRNAs often contain leader sequences that respond to specific metabolites or ions by altering expression of the associated downstream protein-coding sequences. Here we report that the leader RNA of the Mg(2+) transporter gene mgtA of Salmonella enterica, which was previously known to function as a Mg(2+)-sensing riboswitch, harbors an 18 codon proline-rich open reading frame-termed mgtL-that permits intracellular proline to regulate mgtA expression. Interfering with mgtL translation by genetic, pharmacological, or environmental means was observed to increase the mRNA levels from the mgtA coding region. Substitution of the mgtL proline codons by other codons abolished the response to proline and to hyperosmotic stress but not to Mg(2+). Our findings show that mRNA leader sequences can consist of complex regulatory elements that utilize different mechanisms to sense separate signals and mediate an appropriate cellular response.

Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate.

According to the N-end rule, the N-terminal residue of a protein determines its stability. In bacteria, the adaptor ClpS mediates proteolysis by delivering substrates bearing specific N-terminal residues to the protease ClpAP. We now report that the Salmonella adaptor ClpS binds to the N terminus of the regulatory protein PhoP, resulting in PhoP degradation by ClpAP. We establish that the PhoP-activated protein MgtC protects PhoP from degradation by outcompeting ClpS for binding to PhoP. MgtC appears to act exclusively on PhoP, as it did not alter the stability of a different ClpS-dependent ClpAP substrate. Removal of five N-terminal residues rendered PhoP stability independent of both the clpS and mgtC genes. By preserving PhoP protein levels, MgtC enables normal temporal transcription of PhoP-activated genes. The identified mechanism provides a simple means to spare specific substrates from an adaptor-dependent protease.

Advancing evolution: Bacteria break down gene silencer to express horizontally acquired genes.

Horizontal gene transfer advances bacterial evolution. To benefit from horizontally acquired genes, enteric bacteria must overcome silencing caused when the widespread heat-stable nucleoid structuring (H-NS) protein binds to AT-rich horizontally acquired genes. This ability had previously been ascribed to both anti-silencing proteins outcompeting H-NS for binding to AT-rich DNA and RNA polymerase initiating transcription from alternative promoters. However, we now know that pathogenic Salmonella enterica serovar Typhimurium and commensal Escherichia coli break down H-NS when this silencer is not bound to DNA. Curiously, both species use the same protease - Lon - to destroy H-NS in distinct environments. Anti-silencing proteins promote the expression of horizontally acquired genes without binding to them by displacing H-NS from AT-rich DNA, thus leaving H-NS susceptible to proteolysis and decreasing H-NS amounts overall. Conserved amino acid sequences in the Lon protease and H-NS cleavage site suggest that diverse bacteria degrade H-NS to exploit horizontally acquired genes.

Differential synthesis of novel small protein times Salmonella virulence program.

Gene organization in operons enables concerted transcription of functionally related genes and efficient control of cellular processes. Typically, an operon is transcribed as a polycistronic mRNA that is translated into corresponding proteins. Here, we identify a bicistronic operon transcribed as two mRNAs, yet only one allows translation of both genes. We establish that the novel gene ugtS forms an operon with virulence gene ugtL, an activator of the master virulence regulatory system PhoP/PhoQ in Salmonella enterica serovar Typhimurium. Only the longer ugtSugtL mRNA carries the ugtS ribosome binding site and therefore allows ugtS translation. Inside macrophages, the ugtSugtL mRNA species allowing translation of both genes is produced hours before that allowing translation solely of ugtL. The small protein UgtS controls the kinetics of PhoP phosphorylation by antagonizing UgtL activity, preventing premature activation of a critical virulence program. Moreover, S. enterica serovars that infect cold-blooded animals lack ugtS. Our results establish how foreign gene control of ancestral regulators enables pathogens to time their virulence programs.

Degradation of gene silencer is essential for expression of foreign genes and bacterial colonization of the mammalian gut.

Horizontal gene transfer drives bacterial evolution. To confer new properties, horizontally acquired genes must overcome gene silencing by nucleoid-associated proteins, such as the heat-stable nucleoid structuring (H-NS) protein. Enteric bacteria possess proteins that displace H-NS from foreign genes, form nonfunctional oligomers with H-NS, and degrade H-NS, raising the question of whether any of these mechanisms play a role in overcoming foreign gene silencing in vivo. To answer this question, we mutagenized the hns gene and identified a variant specifying an H-NS protein that binds foreign DNA and silences expression of the corresponding genes, like wild-type H-NS, but resists degradation by the Lon protease. Critically, Escherichia coli expressing this variant alone fails to produce curli, which are encoded by foreign genes and required for biofilm formation, and fails to colonize the murine gut. Our findings establish that H-NS proteolysis is a general mechanism of derepressing foreign genes and essential for colonization of mammalian hosts.

Evolution of transcriptional regulatory circuits in bacteria.

Related organisms typically respond to a given cue by altering the level or activity of orthologous transcription factors, which, paradoxically, often regulate expression of distinct gene sets. Although promoter rewiring of shared genes is primarily responsible for regulatory differences among related eukaryotic species, in bacteria, species-specific genes are often controlled by ancestral transcription factors, and regulatory circuit evolution has been further shaped by horizontal gene transfer. Modifications in transcription factors and in promoter structure also contribute to divergence in bacterial regulatory circuits.

Reducing Ribosome Biosynthesis Promotes Translation during Low Mg2+ Stress.

The synthesis of ribosomes is regulated by both amino acid abundance and the availability of ATP, which regenerates guanosine triphosphate (GTP), powers ribosomes, and promotes transcription of rRNA genes. We now report that bacteria supersede both of these controls when experiencing low cytosolic magnesium (Mg2+), a divalent cation essential for ribosome stabilization and for neutralization of ATP's negative charge. We uncover a regulatory circuit that responds to low cytosolic Mg2+ by promoting expression of proteins that import Mg2+ and lower ATP amounts. This response reduces the levels of ATP and ribosomes, making Mg2+ ions available for translation. Mutants defective in Mg2+ uptake and unable to reduce ATP levels accumulate non-functional ribosomal components and undergo translational arrest. Our findings establish a paradigm whereby cells reduce the amounts of translating ribosomes to carry out protein synthesis.

Limitation of phosphate assimilation maintains cytoplasmic magnesium homeostasis.

Phosphorus (P) is an essential component of core biological molecules. In bacteria, P is acquired mainly as inorganic orthophosphate (Pi) and assimilated into adenosine triphosphate (ATP) in the cytoplasm. Although P is essential, excess cytosolic Pi hinders growth. We now report that bacteria limit Pi uptake to avoid disruption of Mg2+-dependent processes that result, in part, from Mg2+ chelation by ATP. We establish that the MgtC protein inhibits uptake of the ATP precursor Pi when Salmonella enterica serovar Typhimurium experiences cytoplasmic Mg2+ starvation. This response prevents ATP accumulation and overproduction of ribosomal RNA that together ultimately hinder bacterial growth and result in loss of viability. Even when cytoplasmic Mg2+ is not limiting, excessive Pi uptake increases ATP synthesis, depletes free cytoplasmic Mg2+, inhibits protein synthesis, and hinders growth. Our results provide a framework to understand the molecular basis for Pi toxicity. Furthermore, they suggest a regulatory logic that governs P assimilation based on its intimate connection to cytoplasmic Mg2+ homeostasis.

RNA chaperone activates Salmonella virulence program during infection.

Organisms often harbor seemingly redundant proteins. In the bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium), the RNA chaperones CspC and CspE appear to play redundant virulence roles because a mutant lacking both chaperones is attenuated, whereas mutants lacking only one exhibit wild-type virulence. We now report that CspC-but not CspE-is necessary to activate the master virulence regulator PhoP when S. Typhimurium experiences mildly acidic pH, such as inside macrophages. This CspC-dependent PhoP activation is specific to mildly acidic pH because a cspC mutant behaves like wild-type S. Typhimurium under other PhoP-activating conditions. Moreover, it is mediated by ugtL, a virulence gene required for PhoP activation inside macrophages. Purified CspC promotes ugtL translation by disrupting a secondary structure in the ugtL mRNA that occludes ugtL's ribosome binding site. Our findings demonstrate that proteins that are seemingly redundant actually confer distinct and critical functions to the lifestyle of an organism.

DNA supercoiling differences in bacteria result from disparate DNA gyrase activation by polyamines.

DNA supercoiling is essential for all living cells because it controls all processes involving DNA. In bacteria, global DNA supercoiling results from the opposing activities of topoisomerase I, which relaxes DNA, and DNA gyrase, which compacts DNA. These enzymes are widely conserved, sharing >91% amino acid identity between the closely related species Escherichia coli and Salmonella enterica serovar Typhimurium. Why, then, do E. coli and Salmonella exhibit different DNA supercoiling when experiencing the same conditions? We now report that this surprising difference reflects disparate activation of their DNA gyrases by the polyamine spermidine and its precursor putrescine. In vitro, Salmonella DNA gyrase activity was sensitive to changes in putrescine concentration within the physiological range, whereas activity of the E. coli enzyme was not. In vivo, putrescine activated the Salmonella DNA gyrase and spermidine the E. coli enzyme. High extracellular Mg2+ decreased DNA supercoiling exclusively in Salmonella by reducing the putrescine concentration. Our results establish the basis for the differences in global DNA supercoiling between E. coli and Salmonella, define a signal transduction pathway regulating DNA supercoiling, and identify potential targets for antibacterial agents.

Salmonella expresses foreign genes during infection by degrading their silencer.

The heat-stable nucleoid structuring (H-NS, also referred to as histone-like nucleoid structuring) protein silences transcription of foreign genes in a variety of Gram-negative bacterial species. To take advantage of the products encoded in foreign genes, bacteria must overcome the silencing effects of H-NS. Because H-NS amounts are believed to remain constant, overcoming gene silencing has largely been ascribed to proteins that outcompete H-NS for binding to AT-rich foreign DNA. However, we report here that the facultative intracellular pathogen Salmonella enterica serovar Typhimurium decreases H-NS amounts 16-fold when inside macrophages. This decrease requires both the protease Lon and the DNA-binding virulence regulator PhoP. The decrease in H-NS abundance reduces H-NS binding to foreign DNA, allowing transcription of foreign genes, including those required for intramacrophage survival. The purified Lon protease degraded free H-NS but not DNA-bound H-NS. By displacing H-NS from DNA, the PhoP protein promoted H-NS proteolysis, thereby de-repressing foreign genes-even those whose regulatory sequences are not bound by PhoP. The uncovered mechanism enables a pathogen to express foreign virulence genes during infection without the need to evolve binding sites for antisilencing proteins at each foreign gene.

The phosphorelay BarA/SirA activates the non-cognate regulator RcsB in Salmonella enterica.

To survive an environmental stress, organisms must detect the stress and mount an appropriate response. One way that bacteria do so is by phosphorelay systems that respond to a stress by activating a regulator that modifies gene expression. To ensure an appropriate response, a given regulator is typically activated solely by its cognate phosphorelay protein(s). However, we now report that the regulator RcsB is activated by both cognate and non-cognate phosphorelay proteins, depending on the condition experienced by the bacterium Salmonella enterica serovar Typhimurium. The RcsC and RcsD proteins form a phosphorelay that activates their cognate regulator RcsB in response to outer membrane stress and cell wall perturbations, conditions Salmonella experiences during infection. Surprisingly, the non-cognate phosphorelay protein BarA activates RcsB during logarithmic growth in Luria-Bertani medium in three ways. That is, BarA's cognate regulator SirA promotes transcription of the rcsDB operon; the SirA-dependent regulatory RNAs CsrB and CsrC further increase RcsB-activated gene transcription; and BarA activates RcsB independently of the RcsC, RcsD, and SirA proteins. Activation of a regulator by multiple sensors broadens the spectrum of environments in which a set of genes is expressed without evolving binding sites for different regulators at each of these genes.

Small proteins regulate Salmonella survival inside macrophages by controlling degradation of a magnesium transporter.

All cells require Mg2+ to replicate and proliferate. The macrophage protein Slc11a1 is proposed to protect mice from invading microbes by causing Mg2+ starvation in host tissues. However, the Mg2+ transporter MgtB enables the facultative intracellular pathogen Salmonella enterica serovar Typhimurium to cause disease in mice harboring a functional Slc11a1 protein. Here, we report that, unexpectedly, the Salmonella small protein MgtR promotes MgtB degradation by the protease FtsH, which raises the question: How does Salmonella preserve MgtB to promote survival inside macrophages? We establish that the Salmonella small protein MgtU prevents MgtB proteolysis, even when MgtR is absent. Like MgtB, MgtU is necessary for survival in Slc11a1+/+ macrophages, resistance to oxidative stress, and growth under Mg2+ limitation conditions. The Salmonella Mg2+ transporter MgtA is not protected by MgtU despite sharing 50% amino acid identity with MgtB and being degraded in an MgtR- and FtsH-dependent manner. Surprisingly, the mgtB, mgtR, and mgtU genes are part of the same transcript, providing a singular example of transcript-specifying proteins that promote and hinder degradation of the same target. Our findings demonstrate that small proteins can confer pathogen survival inside macrophages by altering the abundance of related transporters, thereby furthering homeostasis.

A protein that controls the onset of a Salmonella virulence program.

The mechanism of action and contribution to pathogenesis of many virulence genes are understood. By contrast, little is known about anti-virulence genes, which contribute to the start, progression, and outcome of an infection. We now report how an anti-virulence factor in Salmonella enterica serovar Typhimurium dictates the onset of a genetic program that governs metabolic adaptations and pathogen survival in host tissues. Specifically, we establish that the anti-virulence protein CigR directly restrains the virulence protein MgtC, thereby hindering intramacrophage survival, inhibition of ATP synthesis, stabilization of cytoplasmic pH, and gene transcription by the master virulence regulator PhoP. We determine that, like MgtC, CigR localizes to the bacterial inner membrane and that its C-terminal domain is critical for inhibition of MgtC. As in many toxin/anti-toxin genes implicated in antibiotic tolerance, the mgtC and cigR genes are part of the same mRNA. However, cigR is also transcribed from a constitutive promoter, thereby creating a threshold of CigR protein that the inducible MgtC protein must overcome to initiate a virulence program critical for pathogen persistence in host tissues.