Activation of the P2X7 receptor triggers the elimination of Toxoplasma gondii tachyzoites from infected macrophages

Toxoplasmosis is caused by the protozoan parasite Toxoplasma gondii, which is widespread throughout the world. After active penetration, the parasite is enclosed within a parasitophorous vacuole and survives in the host cell by avoiding, among other mechanisms, lysosomal degradation. A large number of studies have demonstrated the importance of ATP signalling via the P2X₇ receptor, as a component of the inflammatory response against intracellular pathogens. Here we evaluate the effects of extracellular ATP on T. gondii infection of macrophages. ATP treatment inhibits the parasite load and the appearance of large vacuoles in the cytoplasm of intracellular parasites. ROS and NO assays showed that only ROS production is involved with the ATP effects. Immunofluorescence showed colocalization of Lamp1 and SAG1 only after ATP treatment, suggesting the formation of phagolysosomes. The involvement of P2X₇ receptors in T. gondii clearance was confirmed by the use of P2X₇ agonists and antagonists, and by infecting macrophages from P2X₇ receptor-deficient mice. We conclude that parasite elimination might occur following P2X₇ signalling and that novel therapies against intracellular pathogens could take advantage of activation of purinergic signalling.     

Influence of hydrogen acceptors on growth and energy production ofProteus mirabilis

The generation time ofP. mirabilis in defined and in complex medium is shorter in the presence of hydrogen acceptors than in their absence. In the presence of hydrogen acceptors the molar growth yield for glucose and the acetate production are strongly increased. From the molar growth yield and the acetate production Y_ATP in defined medium was calculated as 5.5 g/mole, whereas in complex medium a value of 12.6 g/mole was obtained. The molar growth yield, the acetate production, the amount of hydrogen acceptor reduced and Y_ATP were used to calculate P/2e⁻ratios for phosphorylation coupled to electron transfer to oxygen, nitrate and tetrathionate as respectively 2.80; 1.48 and 1.23 in defined medium. Under anaerobic conditions in the presence of nitrate or tetrathionate as hydrogen acceptor in complex medium a bend in the growth curve is observed. In the period of rapid growth the P/2e⁻ratio for nitrate reduction is of the same magnitude as that in defined medium, however much lower P/2e⁻ratios are found during the subsequent period of slow growth. The P/2e⁻ratios for tetrathionate reduction in complex medium for both growth periods are lower than those in defined medium. Most probably these results indicate that during this period growth and energy production are uncoupled. Under aerobic conditions in complex medium a constant Y_O value of 32.2 g/atom O is found during a short period of the growth curve. Afterwards when the cell density increases a steady decrease of Y_O is observed.     

The P2X7 receptor and intracellular pathogens: a continuing struggle

The purinergic receptor, P2X₇, has recently emerged as an important component of the innate immune response against microbial infections. Ligation of P2X₇ by ATP can stimulate inflammasome activation and secretion of proinflammatory cytokines, but it can also lead directly to killing of intracellular pathogens in infected macrophages and epithelial cells. Thus, while some intracellular pathogens evade host defense responses by modulating with membrane trafficking or cell signaling in the infected cells, the host cells have also developed mechanisms for inhibiting infection. This review will focus on the effects of P2X₇ on control of infection by intracellular pathogens, microbial virulence factors that interfere with P2X₇ activity, and recent evidence linking polymorphisms in human P2X₇ with susceptibility to infection.     

Intracellular sodium homeostasis in relation to the effective free-energy change of ATP hydrolysis: studies of crayfish muscle fibers

We investigated the impact of reductions in the effective free-energy change of ATP hydrolysis (dG/d_ATP) on intracellular sodium homeostasis in bundles of fibers from the abdominal extensor muscle of the crayfish Procambarus clarkii. ³¹P nuclear magnetic resonance (NMR) spectroscopy was used to monitor high-energy phosphate levels and intracellular pH while interleaved ²³Na-NMR spectra were acquired to monitor changes in sodium. Previous work has shown that the bulk of intracellular Na⁺ is NMR visible (see Ivanics et al. 1994). The ²³Na-NMR spectra were diffusion-weighted which effectively filtered out signal contributions from extracellular sodium. The efficacy of this procedure was validated using a relatively non-toxic chemical shift reagent which allowed resolution of extracellular and intracellular Na⁺ signals. Metabolic inhibition (cyanide/iodoacetate) produced pronounced reductions in dG/d_ATP coincident with dramatic increases in intracellular Na⁺ levels ([Na⁺]_i). However, the increases in [Na⁺]_i occurred at dG/d_ATP values well above the threshold value of −46 kJ · mol⁻¹ required by the existing Na⁺ gradient and the membrane potential. These results suggest that the global dG/d_ATP value may not reflect the dG/d_ATP value in the vicinity of the pump. Alternatively, other factors, including low molecular modulators of Na⁺, K⁺-ATPase activity, may be important in this context. Accepted: 15 May 1997     

Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine

In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1α, −6, −7, −8, −18, TNF-α and GM-CSF, but not TGF-β or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen–host cell interactions.     

Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study

The effect of individual environmental conditions (pH, pO₂, temperature, salinity, concentration of ethanol, propanol, tryptone and yeast extract) on the specific growth rate as well as ethanol and glycerol production rate of Saccharomyces cerevisiae S288C was mapped during the fermentative growth in aerobic auxo-accelerostat cultures. The obtained steady-state values of the glycerol to ethanol formation ratio (0.1 mol mol⁻¹) corresponding to those predicted from the stoichiometric model of fermentative yeast growth showed that the complete repression of respiration was obtained in auxostat culture and that the model is suitable for calculation of Y _ATP and Q _ATP values for the aerobic fermentative growth. Smooth decrease in the culture pH and dissolved oxygen concentration (pO₂) down to the critical values of 2.3 and 0.8%, respectively, resulted in decrease in growth yield (Y _ATP) and specific growth rate, however the specific ATP production rate (Q _ATP) stayed almost constant. Increase in the concentration of biomass (>0.8 g dwt l⁻¹), propanol (>2 g l⁻¹) or NaCl (>15 g l⁻¹) lead at first to the decrease in the specific growth rate and Q _ATP, while Y _ATP was affected only at higher concentrations. The observed decrease in Q _ATP was caused by indirect rather than direct inhibition of glycolysis. The increase in tryptone concentration resulted in an increase in the specific growth rate from 0.44 to 0.62 h⁻¹ and Y _ATP from 12.5 to 18.5 mol ATP g dwt⁻¹. This study demonstrates that the auxo-accelerostat method, besides being an efficient tool for obtaining the culture characteristics, provides also decent conditions for the experiments elucidating the control mechanisms of cell growth.     

Excitatory effect of ATP on rat area postrema neurons

ATP-induced inward currents and increases in the cytosolic Ca²⁺ concentration ([Ca]_in) were investigated in neurons acutely dissociated from rat area postrema using whole-cell patch-clamp recordings and fura-2 microfluorometry, respectively. The ATP-induced current (I _ATP) and [Ca]_in increases were mimicked by 2-methylthio-ATP and ATP-γS, and were inhibited by P2X receptor (P2XR) antagonists. The current–voltage relationship of the I _ATP exhibited a strong inward rectification, and the amplitude of the I _ATP was concentration-dependent. The I _ATP was markedly reduced in the absence of external Na⁺, and the addition of Ca²⁺ to Na⁺-free saline increased the I _ATP. ATP did not increase [Ca]_in in the absence of external Ca²⁺, and Ca²⁺ channel antagonists partially inhibited the ATP-induced [Ca]_in increase, indicating that ATP increases [Ca]_in by Ca²⁺ influx through both P2XR channels and voltage-dependent Ca²⁺ channels. There was a negative interaction between P2XR- and nicotinic ACh receptor (nAChR)-channels, which depended on the amplitude and direction of current flow through either channel. Current occlusion was observed at V _hs between −70 and −10 mV when the I _ATP and ACh-induced current (I _ACh) were inward, but no occlusion was observed when these currents were outward at a V _h of +40 mV. The I _ATP was not inhibited by co-application of ACh when the I _ACh was markedly decreased either by removal of permeant cations, by setting V _h close to the equilibrium potential of I _ACh, or by the addition of d-tubocurarine or serotonin. These results suggest that the inhibitory interaction is attributable to inward current flow of cations through the activated P2XR- and nAChR-channels.     

Activation of ATP‐sensitive potassium channels facilitates the function of human endothelial colony‐forming cells via Ca2+/Akt/eNOS pathway

Accumulating data, including those from our laboratory, have shown that the opening of ATP‐sensitive potassium channels (K_ATP) plays a protective role in pulmonary vascular diseases (PVD). As maintainers of the endothelial framework, endothelial colony‐forming cells (ECFCs) are considered excellent candidates for vascular regeneration in cases of PVD. Although K_ATP openers (KCOs) have been demonstrated to have beneficial effects on endothelial cells, the impact of K_ATP on ECFC function remains unclear. Herein, this study investigated whether there is a distribution of K_ATP in ECFCs and what role K_ATP play in ECFC modulation. By human ECFCs isolated from adult peripheral blood, K_ATP were confirmed for the first time to express in ECFCs, comprised subunits of Kir (Kir6.1, Kir6.2) and SUR2b. KCOs such as the classical agent nicorandil (Nico) and the novel agent iptakalim (Ipt) notably improved the function of ECFCs, promoting cell proliferation, migration and angiogenesis, which were abolished by a non‐selective K_ATP blocker glibenclamide (Gli). To determine the underlying mechanisms, we investigated the impacts of KCOs on CaMKII, Akt and endothelial nitric oxide synthase pathways. Enhanced levels were detected by western blotting, which were abrogated by Gli. This suggested an involvement of Ca²⁺ signalling in the regulation of ECFCs by K_ATP. Our findings demonstrated for the first time that there is a distribution of K_ATP in ECFCs and K_ATP play a vital role in ECFC function. The present work highlighted a novel profile of K_ATP as a potential target for ECFC modulation, which may hold the key to the treatment of PVD.     

A novel co‐infection model with Toxoplasma and Chlamydia trachomatis highlights the importance of host cell manipulation for nutrient scavenging

Toxoplasma and Chlamydia trachomatis are obligate intracellular pathogens that have evolved analogous strategies to replicate within mammalian cells. Both pathogens are known to extensively remodel the cytoskeleton, and to recruit endocytic and exocytic organelles to their respective vacuoles. However, how important these activities are for infectivity by either pathogen remains elusive. Here, we have developed a novel co‐infection system to gain insights into the developmental cycles of Toxoplasma and C. trachomatis by infecting human cells with both pathogens, and examining their respective ability to replicate and scavenge nutrients. We hypothesize that the common strategies used by Toxoplasma and Chlamydia to achieve development results in direct competition of the two pathogens for the same pool of nutrients. We show that a single human cell can harbour Chlamydia and Toxoplasma. In co‐infected cells, Toxoplasma is able to divert the content of host organelles, such as cholesterol. Consequently, the infectious cycle of Toxoplasma progresses unimpeded. In contrast, Chlamydia's ability to scavenge selected nutrients is diminished, and the bacterium shifts to a stress‐induced persistent growth. Parasite killing engenders an ordered return to normal chlamydial development. We demonstrate that C. trachomatisenters a stress‐induced persistence phenotype as a direct result from being barred from its normal nutrient supplies as addition of excess nutrients, e.g. amino acids, leads to substantial recovery of Chlamydia growth and infectivity. Co‐infection of C. trachomatis with slow growing strains of Toxoplasma or a mutant impaired in nutrient acquisition does not restrict chlamydial development. Conversely, Toxoplasma growth is halted in cells infected with the highly virulent Chlamydia psittaci. This study illustrates the key role that cellular remodelling plays in the exploitation of host intracellular resources by Toxoplasma and Chlamydia. It further highlights the delicate balance between success and failure of infection by intracellular pathogens in a co‐infection system at the cellular level.     

Modeling K(ATP) channel gating and its regulation

ATP-sensitive potassium (K_ATP) channels couple cell metabolism to plasmalemmal potassium fluxes in a variety of cell types. The activity of these channels is primarily determined by intracellular adenosine nucleotides, which have both inhibitory and stimulatory effects. The role of K_ATP channels has been studied most extensively in pancreatic beta-cells, where they link glucose metabolism to insulin secretion. Many mutations in K_ATP channel subunits (Kir6.2, SUR1) have been identified that cause either neonatal diabetes or congenital hyperinsulinism. Thus, a mechanistic understanding of K_ATP channel behavior is necessary for modeling beta-cell electrical activity and insulin release in both health and disease. Here, we review recent advances in the K_ATP channel structure and function. We focus on the molecular mechanisms of K_ATP channel gating by adenosine nucleotides, phospholipids and sulphonylureas and consider the advantages and limitations of various mathematical models of macroscopic and single-channel K_ATP currents. Finally, we outline future directions for the development of more realistic models of K_ATP channel gating.     

Influence of ATP-dependent K<Superscript>+</Superscript>-channel opener on K<Superscript>+</Superscript>-cycle and oxygen consumption in rat liver mitochondria

The influence of the K_ATP⁺-channel opener diazoxide on the K⁺ cycle and oxygen consumption has been studied in rat liver mitochondria. It was found that diazoxide activates the K_ATP⁺-channel in the range of nanomolar concentrations (50–300 nM, K _1/2 ∼ 140 nM), which results in activation of K⁺/H⁺ exchange in mitochondria. The latter, in turn, accelerates mitochondrial respiration in respiratory state 2. The contribution of K_ATP⁺-channel to the mitochondrial potassium cycle was estimated using the selective K_ATP⁺-channel blocker glibenclamide. The data show that the relative contribution of K_ATP⁺-channel in the potassium cycle of mitochondria is variable and increases only with the decrease in the ATP-independent component of K⁺ uptake. Possible mechanisms underlying the observed phenomena are discussed. The experimental results more fully elucidate the role of K_ATP⁺-channel in the regulation of mitochondrial functions, especially under pathological conditions accompanied by impairment of the mitochondrial energy state.     

Energy conservation during nitrate respiration in Paracoccus denitrificans

P/2e ratios were calculated from anaerobic chemostat cultures of Paracoccus denitrificans with nitrogenous oxides as electron acceptor. P/2e ratios were calculated, using the Y _ATP ^max values determined for aerobic cultures. When succinate was the carbon and energy source the average P/2e values of the sulphate-and succinate-limited cultures with nitrate as electron acceptor were 0.5 and 0.7, respectively, and of the nitrite-limited culture 0.9. With gluconate as carbon and energy source the average P/2e values of the gluconate-limited with nitrate as electron acceptor and nitrate limited cultures were 0.9 and 1.1, respectively.H⁺/O ratios measured in cells obtained from sulphate-, succinate, nitrite-, gluconate-and nitratelimited cultures yielded respective average values of 3.4, 4.5, 3.5, 4.8 and 6.2 for endogenous substrates. From our data we conclude that sulphate-and nitritelimitation causes the loss of site I phosphorylation. Nitrite has no influence on the maximum growth yield on ATP. We propose that metabolism in heterotrophically grown cells of Paracoccus dentrificans is regulated on the level of phosphorylation in the site I region of the electron transport chain.     

Membrane Potassium Channels and Human Bladder Tumor Cells: II. Growth Properties

These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma (HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and ³H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth at concentrations as low as 1 μm (IC₅₀= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range in which glibenclamide decreased open probability of membrane K_ATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined by flow cytometry using 10⁻⁵ m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G₀/G₁ from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens membrane K_ATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We conclude that the sulfonylurea receptor and the corresponding membrane K_ATP channel are involved in mechanisms controlling HTB-9 cell growth. However, K_ATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle. Received: 12 June 1997/Revised: 21 October 1997     

KATP channels process nucleotide signals in muscle thermogenic response

Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K⁺ (K_ATP) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, K_ATP channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how K_ATP channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the K_ATP channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with K_ATP channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal K_ATP channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of K_ATP channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of K_ATP channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders.     

Why intracellular parasitism need not be a degrading experience for Mycobacterium.

The success of mycobacteria as pathogens hinges on their ability to infect and persist within the macrophages of their host. However, activation of host macrophages by cytokines from a productive cellular immune response can stimulate the cells to kill their resident pathogens. This suggests that the interaction between host cell and microbe is in delicate balance, which can be tipped in favour of either organism. Biochemical analysis of mycobacterial vacuoles has shown them to be integral to the host cell''s recycling endosomal system. As such they show limited acidification and hydrolytic activity despite possession of known lysosomal constituents such as cathepsins D, B and L, and LAMP 1. Even in established infections, they remain dynamic compartments accessible to several plasmalemma-derived constituents. Once the macrophage has been activated by IFN-gamma and TNF-alpha the vacuoles coalesce and acidify. This marks a distinct alteration in vacuole physiology and leads to stasis and death of the mycobacteria. Mycobacteria have developed several strategies to avoid this outcome. Most notably, live bacilli-induce sustained release of IL-6 from infected macrophages. IL-6 blocks the ability of both polyclonal primary T cells and T-cell hybridomas to respond to appropriate stimuli. Such an activity could render the centres of infection foci, such as granulomas, anergic and thus avoid release of macrophage-activating cytokines. This paper discusses both the mechanisms by which mycobacteria try to ensure their success as intracellular pathogens and the relevance of these strategies to the overall understanding of mycobacterial diseases.     

Inhibitory effect of κ/β-carrageenan from red alga <Emphasis Type="Italic">Tichocarpus crinitus</Emphasis> on the development of a potato virus X infection in leaves of <Emphasis Type="Italic">Datura stramonium</Emphasis> L.

The effect of κ/β-carrageenan from red alda Tichocarpus crinitus on the development of a potato virus X (PVX) infection in the leaves of Datura stramonium L. has been studied. The treatment of leaves with carrageenan stimulates a protein synthesis in the cells, causing an increase in the size of nucleoli and in the number of mitochondria and membranes of the rough endoplasmic reticulum. At the same time, such treatment slightly stimulates lytic processes, causing an increase in the number of smooth endoplasmic reticulum cisternae, dictyosomes, and cytoplasmic vacuoles and the formation of cytoplasmic electron-transparent zones. The carrageenan-induced stimulation of lytic processes results in the destruction of viral particles and can be considered as one of the defense mechanisms, preventing the intracellular accumulation of virus. The carrageenan-stimulated formation of PVX-specific laminar structures, able to bind viral particles and, therefore, prevent their intracellular translocation and reproduction, represents another carrageenan-induced mechanism of the antiviral defense in plant cells.     

Heme sensing and trafficking in fungi

Fungal pathogens cause life-threatening diseases in humans, and the increasing prevalence of these diseases emphasizes the need for new targets for therapeutic intervention. Nutrient acquisition during infection is a promising target, and recent studies highlight the contributions of endomembrane trafficking, mitochondria, and vacuoles in the sensing and acquisition of heme by fungi. These studies have been facilitated by genetically encoded biosensors and other tools to quantitate heme in subcellular compartments and to investigate the dynamics of trafficking in living cells. In particular, the applications of biosensors in fungi have been extended beyond the detection of metabolites, cofactors, pH, and redox status to include the detection of heme. Here, we focus on studies that make use of biosensors to examine mechanisms of heme uptake and degradation, with guidance from the model fungus Saccharomyces cerevisiae and an emphasis on the pathogenic fungi Candida albicans and Cryptococcus neoformans that threaten human health. These studies emphasize a role for endocytosis in heme uptake, and highlight membrane contact sites involving mitochondria, the endoplasmic reticulum and vacuoles as mediators of intracellular iron and heme trafficking.     

Opening of Cardiac Sarcolemmal KATP Channels by Dinitrophenol Separate from Metabolic Inhibition

Opening of ATP-sensitive K⁺ (K_ATP) channels by the uncoupler of oxidative phosphorylation, 2,4 dinitrophenol (DNP), has been assumed to be secondary to metabolic inhibition and reduced intracellular ATP levels. Herein, we present data which show that DNP (200 μm) can induce opening of cardiac K_ATP channels, under whole-cell and inside-out conditions, despite millimolar concentrations of ATP (1–2.5 mm). DNP-induced currents had a single channel conductance (71 pS), inward rectification, reversal potential, and intraburst kinetic properties (open time constant, τ_open: 4.8 msec; fast closed time constant, τ_closed(f): 0.33 msec) characteristic of K_ATP channels suggesting that DNP did not affect the pore region of the channel, but may have altered the functional coupling of the ATP-dependent channel gating. A DNP analogue, with the pH-titrable hydroxyl replaced by a methyl group, could not open K_ATP channels. The pH-dependence of the effect of DNP on channel opening under whole-cell, cell-attached, and inside-out conditions suggested that transfer of protonated DNP across the sarcolemma is essential for activation of K_ATP channels in the presence of ATP. We conclude that the use of DNP for metabolic stress-induced K_ATP channel opening should be reevaluated. Received: 10 September 1996/Revised: 27 December 1996     

Relaxed natural selection alone does not permit transposable element expansion within 4,000 generations in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Insertion sequences (ISs) are transposable genetic elements in bacterial genomes. IS elements are common among bacteria but are generally rare within free-living species, probably because of the negative fitness effects they have on their hosts. Conversely, ISs frequently proliferate in intracellular symbionts and pathogens that recently transitioned from a free-living lifestyle. IS elements can profoundly influence the genomic evolution of their bacterial hosts, although it is unknown why they often expand in intracellular bacteria. We designed a laboratory evolution experiment with Escherichia coli K-12 to test the hypotheses that IS elements often expand in intracellular bacteria because of relaxed natural selection due to (1) their generally small effective population sizes (N _e) and thus enhanced genetic drift, and (2) their nutrient rich environment, which makes many biosynthetic genes unnecessary and thus selectively neutral territory for IS insertion. We propagated 12 populations under four experimental conditions: large N _e versus small N _e, and nutrient rich medium versus minimal medium. We found that relaxed selection over 4,000 generations was not sufficient to permit IS element expansion in any experimental population, thus leading us to hypothesize that IS expansion in intracellular symbionts may often be spurred by enhanced transposition rates, possibly due to environmental stress, coupled with relaxed natural selection.