Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth.

Salmonella enterica serovar Typhimurium (S. Typhimurium) controls lipopolysaccharide (LPS) biosynthesis by regulating proteolysis of LpxC, the rate-limiting enzyme and target of preclinical antibiotics. PbgA/YejM/LapC regulates LpxC levels and controls outer membrane (OM) LPS composition at the log-to-stationary phase transition. Suppressor substitutions in LPS assembly protein B (LapB/YciM) rescue the LPS and OM integrity defects of pbgA-mutant S. Typhimurium. We hypothesized that PbgA regulates LpxC proteolysis by controlling LapB's ability to bind LpxC as a function of the growth phase. According to existing models, when nutrients are abundant, PbgA binds and restricts LapB from interacting with LpxC and FtsH, which limits LpxC proteolysis. However, when nutrients are limited, there is debate whether LapB dissociates from PbgA to bind LpxC and FtsH to enhance degradation. We sought to examine these models and investigate how the structure of LapB enables salmonellae to control LpxC proteolysis and LPS biosynthesis. Salmonellae increase LapB levels during the stationary phase to promote LpxC degradation, which limits lipid A-core production and increases their survival. The deletion of lapB, resulting in unregulated lipid A-core production and LpxC overabundance, leads to bacterial growth retardation. Tetratricopeptide repeats near the cytosol-inner membrane interface are sufficient for LapB to bind LpxC, and remarkably, LapB and PbgA interact in both growth phases, yet LpxC only associates with LapB in the stationary phase. Our findings support that PbgA-LapB exists as a constitutive complex in S. Typhimurium, which differentially binds LpxC to control LpxC proteolysis and limit lipid A-core biosynthesis in response to changes in the environment.IMPORTANCEAntimicrobial resistance has been a costly setback for human health and agriculture. Continued pursuit of new antibiotics and targets is imperative, and an improved understanding of existing ones is necessary. LpxC is an essential target of preclinical trial antibiotics that can eliminate multidrug-resistant Gram-negative bacterial infections. LapB is a natural LpxC inhibitor that targets LpxC for degradation and limits lipopolysaccharide production in Enterobacteriaceae. Contrary to some studies, findings herein support that LapB remains in complex instead of dissociating from its presumed negative regulator, PbgA/YejM/LapC, under conditions where LpxC proteolysis is enhanced. Advanced comprehension of this critical protein-lipid signaling network will lead to future development and refinement of small molecules that can specifically interfere.

Salmonella enterica Infections Are Disrupted by Two Small Molecules That Accumulate within Phagosomes and Differentially Damage Bacterial Inner Membranes.

Gram-negative bacteria have a robust cell envelope that excludes or expels many antimicrobial agents. However, during infection, host soluble innate immune factors permeabilize the bacterial outer membrane. We identified two small molecules that exploit outer membrane damage to access the bacterial cell. In standard microbiological media, neither compound inhibited bacterial growth nor permeabilized bacterial outer membranes. In contrast, at micromolar concentrations, JAV1 and JAV2 enabled the killing of an intracellular human pathogen, Salmonella enterica serovar Typhimurium. S. Typhimurium is a Gram-negative bacterium that resides within phagosomes of cells from the monocyte lineage. Under broth conditions that destabilized the lipopolysaccharide layer, JAV2 permeabilized the bacterial inner membrane and was rapidly bactericidal. In contrast, JAV1 activity was more subtle: JAV1 increased membrane fluidity, altered reduction potential, and required more time than JAV2 to disrupt the inner membrane barrier and kill bacteria. Both compounds interacted with glycerophospholipids from Escherichia coli total lipid extract-based liposomes. JAV1 preferentially interacted with cardiolipin and partially relied on cardiolipin production for activity, whereas JAV2 generally interacted with lipids and had modest affinity for phosphatidylglycerol. In mammalian cells, neither compound significantly altered mitochondrial membrane potential at concentrations that killed S. Typhimurium. Instead, JAV1 and JAV2 became trapped within acidic compartments, including macrophage phagosomes. Both compounds improved survival of S. Typhimurium-infected Galleria mellonella larvae. Together, these data demonstrate that JAV1 and JAV2 disrupt bacterial inner membranes by distinct mechanisms and highlight how small, lipophilic, amine-substituted molecules can exploit host soluble innate immunity to facilitate the killing of intravesicular pathogens. IMPORTANCE Innovative strategies for developing new antimicrobials are needed. Combining our knowledge of host-pathogen interactions and relevant drug characteristics has the potential to reveal new approaches to treating infection. We identified two compounds with antibacterial activity specific to infection and with limited host cell toxicity. These compounds appeared to exploit host innate immunity to access the bacterium and differentially damage the bacterial inner membrane. Further, both compounds accumulated within Salmonella-containing and other acidic vesicles, a process known as lysosomal trapping, which protects the host and harms the pathogen. The compounds also increased host survival in an insect infection model. This work highlights the ability of host innate immunity to enable small molecules to act as antibiotics and demonstrates the feasibility of antimicrobial targeting of the inner membrane. Additionally, this study features the potential use of lysosomal trapping to enhance the activities of compounds against intravesicular pathogens.

Cardiolipin Biosynthesis Genes Are Not Required for Salmonella enterica Serovar Typhimurium Pathogenesis in C57BL/6J Mice.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that parasitizes macrophages from within a vacuole. The vacuolar environment prompts the bacterium to regulate the lipid composition of the outer membrane (OM), and this influences host inflammation. S. Typhimurium regulates the levels of acidic glycerophospholipids known as cardiolipins (CL) within the OM, and mitochondrial CL molecules can prime and activate host inflammasomes. However, the contribution of S. Typhimurium's CL biosynthesis genes to intracellular survival, inflammasome activation, and pathogenesis had not been examined. S. Typhimurium genes encode three CL synthases. Single, double, and triple mutants were constructed. Similar to other Enterobacteriaceae, ClsA is the primary CL synthase for S. Typhimurium during logarithmic growth, while ClsB and ClsC contribute CL production in stationary phase. It was necessary to delete all three genes to diminish the CL content of the envelope. Despite being devoid of CL molecules, ΔclsABC mutants were highly virulent during oral and systemic infection for C57BL/6J mice. In macrophages, ΔclsA, ΔclsB, ΔclsC, and ΔclsAC mutants behaved like the wild type, whereas ΔclsAB, ΔclsBC, and ΔclsABC mutants were attenuated and elicited reduced amounts of secreted interleukin-1 beta (IL-1ß), IL-18, and lactate dehydrogenase. Hence, when clsA and clsC are deleted, clsB is necessary and sufficient to promote intracellular survival and inflammasome activation. Similarly, when clsB is deleted, clsA and clsC are necessary and sufficient. Therefore, the three CL synthase genes cooperatively and redundantly influence S. Typhimurium inflammasome activation and intracellular survival in C57BL/6J mouse macrophages but are dispensable for virulence in mice. IMPORTANCE Salmonella enterica serovar Typhimurium is a pathogenic Gram-negative bacterium that regulates the cardiolipin (CL) and lipopolysaccharide (LPS) composition of the outer membrane (OM) during infection. Mitochondrial CL molecules activate the inflammasome and its effector caspase-1, which initiates an inflammatory process called pyroptosis. Purified bacterial CL molecules also influence LPS activation of Toll-like receptor 4 (Tlr4). S. Typhimurium resides within macrophage vacuoles and activates Tlr4 and the inflammasome during infection. However, the contribution of the three bacterial CL synthase genes (cls) to microbial pathogenesis and inflammation had not been tested. This study supports that the genes encoding the CL synthases work coordinately to promote intracellular survival in macrophages and to activate the inflammasome but do not influence inflammatory cytokine production downstream of Tlr4 or virulence in C57BL/6J mice. The macrophage phenotypes are not directly attributable to CL production but are caused by deleting specific combinations of cls gene products.

Conserved Tandem Arginines for PbgA/YejM Allow Salmonella Typhimurium To Regulate LpxC and Control Lipopolysaccharide Biogenesis during Infection.

Enterobacteriaceae use the periplasmic domain of the conserved inner membrane protein, PbgA/YejM, to regulate lipopolysaccharide (LPS) biogenesis. Salmonella enterica serovar Typhimurium (S. Typhimurium) relies on PbgA to cause systemic disease in mice and this involves functional interactions with LapB/YciM, FtsH, and LpxC. Escherichia coli PbgA interacts with LapB, an adaptor for the FtsH protease, via the transmembrane segments. LapB and FtsH control proteolysis of LpxC, the rate-limiting LPS biosynthesis enzyme. Lipid A-core, the hydrophobic anchor of LPS molecules, co-crystallizes with PbgA and interacts with residues in the basic region. The model predicts that PbgA-LapB detects periplasmic LPS molecules and prompts FtsH to degrade LpxC. However, the key residues and critical interactions are not defined. We establish that S. Typhimurium uses PbgA to regulate LpxC and define the contribution of two pairs of arginines within the basic region. PbgA R215 R216 form contacts with lipid A-core in the structure, and R231 R232 exist in an adjacent alpha helix. PbgA R215 R216 are necessary for S. Typhimurium to regulate LpxC, control lipid-A core biogenesis, promote survival in macrophages, and enhance virulence in mice. In contrast, PbgA R231 R232 are not necessary to regulate LpxC or to control lipid A-core levels, nor are they necessary to promote survival in macrophages or mice. However, residues R231 R232 are critical for infection lethality, and the persistent infection phenotype requires mouse Toll-like receptor four, which detects lipid A. Therefore, S. Typhimurium relies on PbgA's tandem arginines for multiple interconnected mechanisms of LPS regulation that enhance pathogenesis.

Modulating Isoprenoid Biosynthesis Increases Lipooligosaccharides and Restores Acinetobacter baumannii Resistance to Host and Antibiotic Stress.

Acinetobacter baumannii is a leading cause of ventilator-associated pneumonia and a critical threat due to multidrug resistance. The A. baumannii outer membrane is an asymmetric lipid bilayer composed of inner leaflet glycerophospholipids and outer leaflet lipooligosaccharides. Deleting mlaF of the maintenance of lipid asymmetry (Mla) system causes A. baumannii to become more susceptible to pulmonary surfactants and antibiotics and decreases bacterial survival in the lungs of mice. Spontaneous suppressor mutants isolated from infected mice contain an ISAba11 insertion upstream of the ispB initiation codon, an essential isoprenoid biosynthesis gene. The insertion restores antimicrobial resistance and virulence to ΔmlaF. The suppressor strain increases lipooligosaccharides, suggesting that the mechanism involves balancing the glycerophospholipids/lipooligosaccharides ratio on the bacterial surface. An identical insertion exists in an extensively drug-resistant A. baumannii isolate, demonstrating its clinical relevance. These data show that the stresses bacteria encounter during infection select for genomic rearrangements that increase resistance to antimicrobials.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii.

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism's OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Outer Membrane Lipid Secretion and the Innate Immune Response to Gram-Negative Bacteria.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that consists of inner leaflet phospholipids and outer leaflet lipopolysaccharides (LPS). The asymmetric character and unique biochemistry of LPS molecules contribute to the OM's ability to function as a molecular permeability barrier that protects the bacterium against hazards in the environment. Assembly and regulation of the OM have been extensively studied for understanding mechanisms of antibiotic resistance and bacterial defense against host immunity; however, there is little knowledge on how Gram-negative bacteria release their OMs into their environment to manipulate their hosts. Discoveries in bacterial lipid trafficking, OM lipid homeostasis, and host recognition of microbial patterns have shed new light on how microbes secrete OM vesicles (OMVs) to influence inflammation, cell death, and disease pathogenesis. Pathogens release OMVs that contain phospholipids, like cardiolipins, and components of LPS molecules, like lipid A endotoxins. These multiacylated lipid amphiphiles are molecular patterns that are differentially detected by host receptors like the Toll-like receptor 4/myeloid differentiation factor 2 complex (TLR4/MD-2), mouse caspase-11, and human caspases 4 and 5. We discuss how lipid ligands on OMVs engage these pattern recognition receptors on the membranes and in the cytosol of mammalian cells. We then detail how bacteria regulate OM lipid asymmetry, negative membrane curvature, and the phospholipid-to-LPS ratio to control OMV formation. The goal is to highlight intersections between OM lipid regulation and host immunity and to provide working models for how bacterial lipids influence vesicle formation.

Salmonella enterica Serovar Typhimurium Uses PbgA/YejM To Regulate Lipopolysaccharide Assembly during Bacteremia.

Salmonella enterica serovar Typhimurium (S Typhimurium) relies upon the inner membrane protein PbgA to enhance outer membrane (OM) integrity and promote virulence in mice. The PbgA transmembrane domain (residues 1 to 190) is essential for viability, while the periplasmic domain (residues 191 to 586) is dispensable. Residues within the basic region (residues 191 to 245) bind acidic phosphates on polar phospholipids, like for cardiolipins, and are necessary for salmonella OM integrity. S Typhimurium bacteria increase their OM cardiolipin concentrations during activation of the PhoPQ regulators. The mechanism involves PbgA's periplasmic globular region (residues 245 to 586), but the biological role of increasing cardiolipins on the surface is not understood. Nonsynonymous polymorphisms in three essential lipopolysaccharide (LPS) synthesis regulators, lapB (also known as yciM), ftsH, and lpxC, variably suppressed the defects in OM integrity, rifampin resistance, survival in macrophages, and systemic colonization of mice in the pbgAΔ191-586 mutant (in which the PbgA periplasmic domain from residues 191 to 586 is deleted). Compared to the OMs of the wild-type salmonellae, the OMs of the pbgA mutants had increased levels of lipid A-core molecules, cardiolipins, and phosphatidylethanolamines and decreased levels of specific phospholipids with cyclopropanated fatty acids. Complementation and substitution mutations in LapB and LpxC generally restored the phospholipid and LPS assembly defects for the pbgA mutants. During bacteremia, mice infected with the pbgA mutants survived and cleared the bacteria, while animals infected with wild-type salmonellae succumbed within 1 week. Remarkably, wild-type mice survived asymptomatically with pbgA-lpxC salmonellae in their livers and spleens for months, but Toll-like receptor 4-deficient animals succumbed to these infections within roughly 1 week. In summary, S Typhimurium uses PbgA to influence LPS assembly during stress in order to survive, adapt, and proliferate within the host environment.

The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane.

The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened Acinetobacter baumannii transposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

Salmonella Tol-Pal Reduces Outer Membrane Glycerophospholipid Levels for Envelope Homeostasis and Survival during Bacteremia.

Salmonellae regulate membrane lipids during infection, but the exact proteins and mechanisms that promote their survival during bacteremia remain largely unknown. Mutations in genes encoding the conserved Salmonella enterica serovar Typhimurium (S Typhimurium) Tol-Pal apparatus caused the outer membrane (OM) sensor lipoprotein, RcsF, to become activated. The capsule activation phenotype for the mutants suggested that Tol-Pal might influence envelope lipid homeostasis. The mechanism involves reducing OM glycerophospholipid (GPL) levels, since the mutant salmonellae similarly accumulated phosphatidylglycerols (PGl) and phosphatidylethanolamines (PE) within the OM in comparison to the wild type. The data support the Escherichia coli model, whereby Tol-Pal directs retrograde GPL translocation across the periplasm. The S Typhimurium mechanism involves contributions from YbgC, a cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase, and CpoB, a periplasmic TolA-binding protein. The functional relationship between Tol-Pal and YbgC and CpoB was previously unresolved. The S Typhimurium Tol-Pal proteins contribute similarly toward promoting OM-GPL homeostasis and Rcs signaling inactivity but differently toward promoting bacterial morphology, rifampin resistance, survival in macrophages, and survival in mice. For example, tolQ, tolR, tolA, and cpoB mutants were significantly more attenuated than ybgC, tolB, and pal mutants in a systemic mouse model of disease. Therefore, key roles exist for TolQ, TolR, TolA, and CpoB during murine bacteremia, which are independent of maintaining GPL homeostasis. The ability of TolQR to channel protons across the inner membrane (IM) is necessary for S Typhimurium TolQRA function, since mutating conserved channel-facing residues rendered TolQ ineffective at rescuing deletion mutant phenotypes. Therefore, Tol-Pal promotes S Typhimurium survival during bacteremia, in part, by reducing OM GPL concentrations, while TolQRA and CpoB enhance systemic virulence by additional mechanisms.

Cues from the Membrane: Bacterial Glycerophospholipids.

In this issue of the Journal of Bacteriology, V. W. Rowlett et al. unveil new Escherichia coli circuitry linking membrane glycerophospholipid (GPL) homeostasis to bacterial stress response and adaptation mechanisms (J Bacteriol 199:e00849-16, 2017, https://doi.org/10.1128/JB.00849-16). Glycerophospholipids comprise critical components of the dual-membrane envelope of Gram-negative bacteria and participate in many processes. The new evidence suggests that, in some instances, distinct E. coli GPL molecules function for distinct biochemistry and bacteria sense perturbations in membrane GPL concentrations to coordinate survival strategies. Understanding GPL sensing and remodeling mechanisms will be important moving forward, given the breadth of function for these molecules in bacteriology.

Delivery of cardiolipins to the Salmonella outer membrane is necessary for survival within host tissues and virulence.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that serves as a barrier to the environment. During infection, Gram-negative bacteria remodel their OM to promote survival and replication within host tissues. Salmonella rely on the PhoPQ two-component regulators to coordinate OM remodeling in response to environmental cues. In a screen for mediators of PhoPQ-regulated OM remodeling in Salmonella Typhimurium, we identified PbgA, a periplasmic domain-containing transmembrane protein, which binds cardiolipin glycerophospholipids near the inner membrane and promotes their PhoPQ-regulated trafficking to the OM. Purified-PbgA oligomers are tetrameric, and the periplasmic domain contains a globular region that binds to the OM in a PhoPQ-dependent manner. Thus, PbgA forms a complex that may bridge the envelope for regulated cardiolipin delivery. PbgA globular region-deleted mutant bacteria are severely attenuated for pathogenesis, suggesting that increased cardiolipin trafficking to the OM is necessary for Salmonella to survive within host tissues that activate PhoPQ.

Salmonellae PhoPQ regulation of the outer membrane to resist innate immunity.

Salmonellae sense host cues to regulate properties important for bacterial survival and replication within host tissues. The PhoPQ two-component regulatory system senses phagosome acidification and cationic antimicrobial peptides (CAMP) to regulate the protein and lipid contents of the bacterial envelope that comprises an inner and outer membrane. PhoPQ-regulated lipid components of the outer membrane include lipopolysaccharides and glycerophospholipids. Envelope proteins regulated by PhoPQ, include: components of virulence associated secretion systems, the flagellar apparatus, membrane transport systems, and proteins that are likely structural components of the outer membrane. PhoPQ alteration of the bacterial surface results in increased bacterial resistance to CAMP and decreased detection by the innate immune system. This review details the molecular complexity of the bacterial cell envelope and highlights the outer membrane lipid bilayer as an environmentally regulated bacterial organelle.

PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane.

Gram-negative bacteria have two lipid membranes separated by a periplasmic space containing peptidoglycan. The surface bilayer, or outer membrane (OM), provides a barrier to toxic molecules, including host cationic antimicrobial peptides (CAMPs). The OM comprises an outer leaflet of lipid A, the bioactive component of lipopolysaccharide (LPS), and an inner leaflet of glycerophospholipids (GPLs). The structure of lipid A is environmentally regulated in a manner that can promote bacterial infection by increasing bacterial resistance to CAMP and reducing LPS recognition by the innate immune system. The gastrointestinal pathogen, Salmonella Typhimurium, responds to acidic pH and CAMP through the PhoPQ two-component regulatory system, which stimulates lipid A remodeling, CAMP resistance, and intracellular survival within acidified phagosomes. Work here demonstrates that, in addition to regulating lipid A structure, the S. Typhimurium PhoPQ virulence regulators also regulate acidic GPL by increasing the levels of cardiolipins and palmitoylated acylphosphatidylglycerols within the OM. Triacylated palmitoyl-PG species were diminished in strains deleted for the PhoPQ-regulated OM lipid A palmitoyltransferase enzyme, PagP. Purified PagP transferred palmitate to PG consistent with PagP acylation of both lipid A and PG within the OM. Therefore, PhoPQ coordinately regulates OM acidic GPL with lipid A structure, suggesting that GPLs cooperate with lipid A to form an OM barrier critical for CAMP resistance and intracellular survival of S. Typhimurium.

ppGpp: magic beyond RNA polymerase.

During stress, bacteria undergo extensive physiological transformations, many of which are coordinated by ppGpp. Although ppGpp is best known for enhancing cellular resilience by redirecting the RNA polymerase (RNAP) to certain genes, it also acts as a signal in many other cellular processes in bacteria. After a brief overview of ppGpp biosynthesis and its impact on promoter selection by RNAP, we discuss how bacteria exploit ppGpp to modulate the synthesis, stability or activity of proteins or regulatory RNAs that are crucial in challenging environments, using mechanisms beyond the direct regulation of RNAP activity.

ppGpp conjures bacterial virulence.

Like for all microbes, the goal of every pathogen is to survive and replicate. However, to overcome the formidable defenses of their hosts, pathogens are also endowed with traits commonly associated with virulence, such as surface attachment, cell or tissue invasion, and transmission. Numerous pathogens couple their specific virulence pathways with more general adaptations, like stress resistance, by integrating dedicated regulators with global signaling networks. In particular, many of nature's most dreaded bacteria rely on nucleotide alarmones to cue metabolic disturbances and coordinate survival and virulence programs. Here we discuss how components of the stringent response contribute to the virulence of a wide variety of pathogenic bacteria.

Distinct roles of ppGpp and DksA in Legionella pneumophila differentiation.

To transit between hosts, intracellular Legionella pneumophila transform into a motile, infectious, transmissive state. Here we exploit the pathogen's life cycle to examine how guanosine tetraphosphate (ppGpp) and DksA cooperate to govern bacterial differentiation. Transcriptional profiling revealed that during transmission alarmone accumulation increases the mRNA for flagellar and Type IV-secretion components, secreted host effectors and regulators, and decreases transcripts for translation, membrane modification and ATP synthesis machinery. DksA is critical for differentiation, since mutants are defective for stationary phase survival, flagellar gene activation, lysosome avoidance and macrophage cytotoxicity. The roles of ppGpp and DksA depend on the context. For macrophage transmission, ppGpp is essential, whereas DksA is dispensable, indicating that ppGpp can act autonomously. In broth, DksA promotes differentiation when ppGpp levels increase, or during fatty acid stress, as judged by flaA expression and evasion of degradation by macrophages. For flagella morphogenesis, DksA is required for basal fliA (sigma(28)) promoter activity. When alarmone levels increase, DksA cooperates with ppGpp to generate a pulse of Class II rod RNA or to amplify the Class III sigma factor and Class IV flagellin RNAs. Thus, DksA responds to the level of ppGpp and other stress signals to co-ordinate L. pneumophila differentiation.

Legionella pneumophila couples fatty acid flux to microbial differentiation and virulence.

During its life cycle, Legionella pneumophila alternates between at least two phenotypes: a resilient, infectious form equipped for transmission and a replicative cell type that grows in amoebae and macrophages. Considering its versatility, we postulated that multiple cues regulate L. pneumophila differentiation. Beginning with a Biolog Phenotype MicroArray screen, we demonstrate that excess short-chain fatty acids (SCFAs) trigger replicative cells to cease growth and activate their panel of transmissive traits. To co-ordinate their response to SCFAs, L. pneumophila utilizes the LetA/LetS two-component system, but not phosphotransacetylase or acetyl kinase, two enzymes that generate high-energy phosphate intermediates. Instead, the stringent response enzyme SpoT appears to monitor fatty acid biosynthesis to govern transmission trait expression, as an altered distribution of acylated acyl carrier proteins correlated with the SpoT-dependent differentiation of cells treated with either excess SCFAs or the fatty acid biosynthesis inhibitors cerulenin and 5-(tetradecyloxy)-2-furoic acid. We postulate that, by exploiting the stringent response pathway to couple cellular differentiation to its metabolic state, L. pneumophila swiftly acclimates to stresses encountered in its host or the environment, thereby enhancing its overall fitness.

SpoT governs Legionella pneumophila differentiation in host macrophages.

During its life cycle, Legionella pneumophila alternates between a replicative and a transmissive state. To determine their contributions to L. pneumophila differentiation, the two ppGpp synthetases, RelA and SpoT, were disrupted. Synthesis of ppGpp was required for transmission, as relA spoT mutants were killed during entry to and exit from macrophages. RelA, which senses amino acid starvation induced by serine hydroxamate, is dispensable in macrophages, as relA mutants spread efficiently. SpoT monitors fatty acid biosynthesis (FAB), since following cerulenin treatment, wild-type and relA strains expressed the flaA transmissive gene, but relA spoT mutants did not. As in Escherichia coli, the SpoT response to FAB perturbation likely required an interaction with acyl-carrier protein (ACP), as judged by the failure of the spoT-A413E allele to rescue transmissive trait expression of relA spoT bacteria. Furthermore, SpoT was essential for transmission between macrophages, since secondary infections by relA spoT mutants were restored by induction of spoT, but not relA. To resume replication, ppGpp must be degraded, as mutants lacking spoT hydrolase activity failed to convert from the transmissive to the replicative phase in either bacteriological medium or macrophages. Thus, L. pneumophila requires SpoT to monitor FAB and to alternate between replication and transmission in macrophages.

Fungal hydroquinones contribute to brown rot of wood.

The fungi that cause brown rot of wood initiate lignocellulose breakdown with an extracellular Fenton system in which Fe(2+) and H(2)O(2) react to produce hydroxyl radicals (.OH), which then oxidize and cleave the wood holocellulose. One such fungus, Gloeophyllum trabeum, drives Fenton chemistry on defined media by reducing Fe(3+) and O(2) with two extracellular hydroquinones, 2,5-dimethoxyhydroquinone (2,5-DMHQ) and 4,5-dimethoxycatechol (4,5-DMC). However, it has never been shown that the hydroquinones contribute to brown rot of wood. We grew G. trabeum on spruce blocks and found that 2,5-DMHQ and 4,5-DMC were each present in the aqueous phase at concentrations near 20 microM after 1 week. We determined rate constants for the reactions of 2,5-DMHQ and 4,5-DMC with the Fe(3+)-oxalate complexes that predominate in wood undergoing brown rot, finding them to be 43 l mol(-1) s(-1) and 65 l mol(-1) s(-1) respectively. Using these values, we estimated that the average amount of hydroquinone-driven .OH production during the first week of decay was 11.5 micromol g(-1) dry weight of wood. Viscometry of the degraded wood holocellulose coupled with computer modelling showed that a number of the same general magnitude, 41.2 micromol oxidations per gram, was required to account for the depolymerization that occurred in the first week. Moreover, the decrease in holocellulose viscosity was correlated with the measured concentrations of hydroquinones. Therefore, hydroquinone-driven Fenton chemistry is one component of the biodegradative arsenal that G. trabeum expresses on wood.