The freeze-avoiding mountain pine beetle (Dendroctonus ponderosae) survives prolonged exposure to stressful cold by mitigating ionoregulatory collapse.

Insect performance is linked to environmental temperature, and surviving through winter represents a key challenge for temperate, alpine and polar species. To overwinter, insects have adapted a range of strategies to become truly cold hardy. However, although the mechanisms underlying the ability to avoid or tolerate freezing have been well studied, little attention has been given to the challenge of maintaining ion homeostasis at frigid temperatures in these species, despite this limiting cold tolerance for insects susceptible to mild chilling. Here, we investigated how prolonged exposure to temperatures just above the supercooling point affects ion balance in freeze-avoidant mountain pine beetle (Dendroctonus ponderosae) larvae in autumn, mid-winter and spring, and related it to organismal recovery times and survival. Hemolymph ion balance was gradually disrupted during the first day of exposure, characterized by hyperkalemia and hyponatremia, after which a plateau was reached and maintained for the rest of the 7-day experiment. The degree of ionoregulatory collapse correlated strongly with recovery times, which followed a similar asymptotical progression. Mortality increased slightly during extensive cold exposures, where hemolymph K+ concentration was highest, and a sigmoidal relationship was found between survival and hyperkalemia. Thus, the cold tolerance of the freeze-avoiding larvae of D. ponderosae appears limited by the ability to prevent ionoregulatory collapse in a manner similar to that of chill-susceptible insects, albeit at much lower temperatures. Based on these results, we propose that a prerequisite for the evolution of insect freeze avoidance may be a convergent or ancestral ability to maintain ion homeostasis during extreme cold stress.

Synthesis, characterization and assessment of antioxidant and antibacterial potential of dihydroquinoline-3-carboxylic acid and their metal derivatives.

Metal complexes of drug are used to inhibit growth of pathogenic microorganisms and reduces drug resistance. Moxifloxacin is a dihydroquinoline-3-carboxylic acid 4th generation fluoroquinolone antibiotic that has tendency to bind with metal ions. In current study four moxifloxacin-metal complexes i.e. Moxifloxacin-sliver (Moxi-Ag), Moxifloxacin-rhodium (Moxi-Rh), Moxifloxacin-titanium (Moxi-Ti) and Moxifloxacin-rubidium (Moxi-Rb) have been synthesized and evaluated for antibacterial activities against resistant microorganisms along with antioxidant effects. The structure elucidation was carried out using FTIR, 1H- NMR and UV-Vis spectroscopy. Agar well diffusion method and DPPH (1, 1- dipheny1-2-picrylhydrazyl) methods were used to study the antibacterial and antioxidant activity respectively. Both 1H NMR and FTIR spectra clearly showed that Moxi-metal complexes are formed due to change in their carboxyl stretching band in IR, H-2 and H-5 peak position in 1H NMR. All the Moxi-metal complexes showed distinguished antibacterial effects against both Gram-negative and Gram-positive bacteria as compared to drug which was found resistant against many microorganisms. Moxi-Rb and Moxi-Ag metal complexes showed higher antioxidant activity (IC50 values range from 8.26 - 9.19 µg/ml) than Moxi-Ti and Moxi-Rh metal complexes (IC50 range from 11.23 - 14.65 µg/ml).

Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica.

Oxygen fluctuations are common in marine waters, and hypoxia-reoxygenation (H-R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h <0.01% O2) and subsequent reoxygenation (1.5 h 21% O2) compared with normoxic control animals (21% O2). We demonstrated that flux capacity of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased the oxygen flux capacity of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H-R stress, whereas control by the proton leak subsystem of LEAK respiration increased during hypoxia and returned to normoxic levels during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, whereas it was suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.

Salinity-dependent effects of ZnO nanoparticles on bioenergetics and intermediate metabolite homeostasis in a euryhaline marine bivalve, Mytilus edulis.

Engineered nanoparticles including ZnO nanoparticles (nZnO) are important emerging pollutants in aquatic ecosystems creating potential risks to coastal ecosystems and associated biota. The toxicity of nanoparticles and its interaction with the important environmental stressors (such as salinity variation) are not well understood in coastal organisms and require further investigation. Here, we examined the interactive effects of 100 µg l-1 nZnO or dissolved Zn (as a positive control for Zn2+ release) and salinity (normal 15, low 5, and fluctuating 5-15) on bioenergetics and intermediate metabolite homeostasis of a keystone marine bivalve, the blue mussel Mytilus edulis from the Baltic Sea. nZnO exposures did not lead to strong disturbances in energy or intermediate metabolite homeostasis regardless of the salinity regime. Dissolved Zn exposures suppressed the mitochondrial ATP synthesis capacity and coupling as well as anaerobic metabolism and modified the free amino acid profiles in the mussels indicating that dissolved Zn is metabolically more damaging than nZnO. The environmental salinity regime strongly affected metabolic homeostasis and altered physiological and biochemical responses to nZnO or dissolved Zn in the mussels. Exposure to low (5) or fluctuating (5-15) salinity affected the physiological condition, energy metabolism and homeostasis, as well as amino acid metabolism in M. edulis. Generally, fluctuating salinity (5-15) appeared bioenergetically less stressful than constantly hypoosmotic stress (salinity 5) in M. edulis indicating that even short (24 h) periods of recovery might be sufficient to restore the metabolic homeostasis in this euryhaline species. Notably, the biological effects of nZnO and dissolved Zn became progressively less detectable as the salinity stress increased. These findings demonstrate that habitat salinity must be considered in the biomarker-based assessment of the toxic effects of nanopollutants on coastal organisms.

Effects of prolonged food limitation on energy metabolism and burrowing activity of an infaunal marine bivalve, Mya arenaria.

Benthic organisms are subject to prolonged seasonal food limitation in the temperate shallow coastal waters that can cause energetic stress and affect their performance. Sediment-dwelling marine bivalves cope with prolonged food limitation by adjusting different physiological processes that might cause trade-offs between maintenance and other fitness-related functions. We investigated the effects of prolonged (42 days) food deprivation on bioenergetics, burrowing performance and amino acid profiles in a common marine bivalve, Mya arenaria collected in winter and spring. Food limitation of >15 days decreased respiration of the clams by 80%. Total tissue energy content was higher in spring-collected clams (reflecting higher lipid content) than in their winter counterparts. Prolonged food deprivation decreased the tissue energy content of clams, especially in winter. The levels of free amino acids transiently increased during the early phase of food deprivation possibly reflecting suppression of the protein synthesis or enhanced protein degradation. The levels of amino acids considered essential for bivalves were more tightly conserved than those of non-essential amino acids during starvation. The burrowing capacity of clams was negatively affected by food deprivation so that the time required for a burial cycle increased by 35-50% after 22-42 days of starvation. During the early phase of starvation, clams preferentially used lipids as fuel for burrowing, whereas carbohydrates were used at the later phase. These findings suggest that although M. arenaria can withstand prolonged food deprivation by lowering their basal maintenance costs and switching their fuel usage, their ecological functions (e.g. bioturbation and the energy transferable to the next trophic level) could be negatively impacted by starvation.

Evaluation of anti-inflammatory and antibacterial potential of newly synthesized 4-(2-Keto-1-benzimidazollinyl) derivatives of piperidine.

Benzimidazole and its derivatives found variety of biological activities, for the searching of its potent anti-inflammatory analogues, we synthesized four novel 4-(2-keto-1-benzimidazollinyl) piperidine derivatives (Q1 to Q4) by refluxing piperidine with substituted imidazole and subjected to in-vitro anti-inflammatory (ROS, NO) and antibacterial activities, structures were elucidated using spectroscopic techniques. Results revealed that compound Q1 showed most effective anti-inflammatory activity with IC 50 7.6±1.3 µg/ml compared with standard Ibuprofen having IC50 11.2±1.9µg/mL. Compound Q3 showed good activity for Nitrite accumulation by stimulating macrophages test similar to standard NG Methyl L-arginine acetate with IC50 value 24.2±0.8µg/mL. The antibacterial activity of these compounds were evaluated against selected Gram+ve E. faecalis, C. diphtheriae, S. aureus and Gram -ve organism E. coli, Enterobacter aerogenes and P. aeruginosa. Synthesized compounds showed low to moderate level of antibacterial activity Q1 showed the highest antibacterial activity against Enterococcus faecalis and Escherichia coli with zone of inhibition 18mm and Q3 showed highest activity against Corynebacterium diptheriae (ZOI:18mm). Structure-activity relationship (SAR) study revealed that among all the synthesized compounds unsubstituted naphthalene (Q1) and phenyl (Q3) ring containing derivatives were most potent.

Effects of hypoxia and reoxygenation on intermediary metabolite homeostasis of marine bivalves Mytilus edulis and Crassostrea gigas.

Marine benthic invertebrates are frequently exposed to fluctuating oxygen levels resulting in hypoxia-reoxygenation (H/R) stress in the intertidal, estuarine and shallow coastal habitats. H/R stress can strongly affect the organisms' physiological performance due to the negative shifts in bioenergetics and redox balance. H/R stress commonly leads to the depletion of energy substrates and accumulation of anaerobic end products, but the effects of H/R stress on the homeostasis of the intermediate nitrogenous compounds are not well understood. We studied the effects of the short-term and long-term hypoxia (1 and 6 days, respectively) and subsequent reoxygenation on the metabolite profiles of free amino acids (FAAs), as well as the intermediates of the urea cycle and purine metabolism in two species of hypoxia-tolerant intertidal bivalves, the blue mussels Mytilus edulis and the Pacific oysters Crassostrea gigas. Accumulation of succinate was assessed to determine the role of anaerobiosis in the metabolic responses to H/R stress. Our study showed that the more hypoxia-tolerant of the two studied species (C. gigas) had lower rate of succinate accumulation during hypoxia (indicating stronger metabolic rate suppression) and was better able to maintain the homeostasis of nitrogenous intermediates during H/R stress compared with the less hypoxia-tolerant M. edulis. Furthermore, analysis of the metabolite profiles indicate that the oysters maintain high levels of cytoprotective compounds (such as taurine and GABA), accumulate lower levels of potential prooxidants (such as succinate and hypoxanthine) and experience less damage to oxidation-prone thiol-containing amino acids such as cysteine, homocysteine and methionine during hypoxia and reoxygenation compared with the blue mussels. This study indicates a potentially important role of intermediate metabolite homeostasis in the tolerance to prolonged hypoxia and H/R stress in marine organisms and opens avenue for further testing of this hypothesis in a broader comparative framework.

Persistence and safety assessment of novel probiotic strain Lactobacillus plantarum 1 strain Lp86 and Lp36 in Salmonella typhi infected mice.

The species of Lactic acid bacteria are known to confer beneficial effects on the host by inhabiting in their gastrointestinal tract (GIT). They succeed in surviving the harsh conditions of the GIT by exhibiting strong tolerance against gastric acids, digestive enzymes and bile simultaneously antagonizing the pathogens by production of antimicrobials. This study has been conducted to elaborate these probiotic characteristics in vivo for which mice were intragastrically given a probiotic approved dose of 1011cfu/ml for 4 days to assess the persistence of two probiotic candidates Lactobacillus plantarum Lp36 and Lactobacillus plantarum Lp86. The fecal count of the test probiotic candidates were seen to persist well in the GIT for 15 days with a count ranging between 104-108cfu/ mg of feces (p>0.01). The safety assessment of L. plantarum Lp36 in healthy and S. typhi in infected mice showed an increase in cell count from (day zero of inoculation) 106cfu/100mg of feces to108cfu/100mg (p>0.01) which was maintained till day six, suggesting the persistence in the GIT. The sections of the mice intestinal lining under scanning electron microscope revealed the adherence of Lp36 and Lp86 to the intestinal epithelia. The mice did not show any adverse effect on its health. These findings make our strains promising probiotic candidates to be used to promote health benefits after further assessments.

Mitochondrial Mechanisms Underlying Tolerance to Fluctuating Oxygen Conditions: Lessons from Hypoxia-Tolerant Organisms.

Oxygen (O2) is essential for most metazoan life due to its central role in mitochondrial oxidative phosphorylation (OXPHOS), which generates >90% of the cellular adenosine triphosphate. O2 fluctuations are an ultimate mitochondrial stressor resulting in mitochondrial damage, energy deficiency, and cell death. This work provides an overview of the known and putative mechanisms involved in mitochondrial tolerance to fluctuating O2 conditions in hypoxia-tolerant organisms including aquatic and terrestrial vertebrates and invertebrates. Mechanisms of regulation of the mitochondrial OXPHOS and electron transport system (ETS) (including alternative oxidases), sulphide tolerance, regulation of redox status and mitochondrial quality control, and the potential role of hypoxia-inducible factor (HIF) in mitochondrial tolerance to hypoxia are discussed. Mitochondrial phenotypes of distantly related animal species reveal common features including conservation and/or anticipatory upregulation of ETS capacity, suppression of reactive oxygen species (ROS)-producing electron flux through ubiquinone, reversible suppression of OXPHOS activity, and investment into the mitochondrial quality control mechanisms. Despite the putative importance of oxidative stress in adaptations to hypoxia, establishing the link between hypoxia tolerance and mitochondrial redox mechanisms is complicated by the difficulties of establishing the species-specific concentration thresholds above which the damaging effects of ROS outweigh their potentially adaptive signaling function. The key gaps in our knowledge about the potential mechanisms of mitochondrial tolerance to hypoxia include regulation of mitochondrial biogenesis and fusion/fission dynamics, and HIF-dependent metabolic regulation that require further investigation in hypoxia-tolerant species. Future physiological, molecular and genetic studies of mitochondrial responses to hypoxia, and reoxygenation in phylogenetically diverse hypoxia-tolerant species could reveal novel solutions to the ubiquitous and metabolically severe problem of O2 deficiency and would have important implications for understanding the evolution of hypoxia tolerance and the potential mitigation of pathological states caused by O2 fluctuations.

Probiotic potential of novel strains of Lactobacillus plantarum Lp -1: In vitro studies.

Five Lactobacillus strains isolated from vegetable and dairy products showed 99% similarity with Lactobacillus plantarum 1(Lp-1) using API -CHL 50 kit. Most of them proved to be sensitive to bacterial cell-wall inhibitors i.e. penicillin, ampicillin, amoxicillin and methicillin as studied by disc-diffusion method. These strains manifested profound tolerance to acidic-stress where Lp86 and Lp36 exhibited a good survival pattern at pH-2 for 4 hr retaining a survival count of 85% and 50%, respectively. A high survival of 85.7% was witnessed in Lp86 in presence of protease while Lp36 maintained 94.55% and 92.65% of population under the influence of enzyme pancreatin and pepsin. All the strains displayed marked tolerance against trypsin as the count did not drop below 77%. Absorbance and growth in terms of cfu/ml for bile-tolerance was examined for concentrations reflecting those in the GIT of humans, all the Lp-1 strains when grown with 1% bile showed a drop in the viable count by 1 log cycle i.e. from 1010 to 109cfu/ml. Fulfilling the above mentioned criteria these probiotic candidates displayed their capacity to reach the colon as viable metabolically active cells after successfully surviving under conditions similar to the gastrointestinal tract of humans. Upon examining the viability and stability of these probiotic candidates in most common foods serving as vehicle for probiotic delivery to the intestine, it was noticed that all the isolates tested sustained a probiotic approved number of 107 cfu/ml for effective function as recommended by WHO, after a maximum storage for one month. Hence, it could be justified that the selected probiotic candidates possess prominent probiotic potential. Therefore, L. plantarum 1 strains could prove to be an efficient probiotic after further in vivo studies to explore its safety in human subjects.

Effects of a common pharmaceutical, atorvastatin, on energy metabolism and detoxification mechanisms of a marine bivalve Mytilus edulis.

Biologically active compounds from pharmaceuticals cause concern due to their common occurrence in water and sediments of urbanized coasts and potential threat to marine organisms. Atorvastatin (ATO), a globally prescribed drug, is environmentally stable and bioavailable to marine organisms; however, the physiological and toxic effects of this drug on ecologically important coastal species are yet to be elucidated. We studied the effect of ATO (˜1.2 µg L-1) on bioenergetics (including whole-organism and mitochondrial respiration, as well as tissue energy reserves and mRNA expression of genes involved in mitochondrial biogenesis and fatty acid metabolism in the gills and the digestive gland) of a keystone bivalve Mytulis edulis (the blue mussel) from the Baltic Sea. Xenobiotic detoxification systems including activity and mRNA expression of P-glycoprotein, and Phase I and II biotransformation enzymes (cytochrome P450 monooxygenase CYP1A and glutathione transferase, GST) were also assessed in the gill and digestive gland of the mussels. Exposure to ATO caused rapid uptake and biotransformation of the drug by the mussels. Standard metabolic rate of ATO-exposed mussels increased by 56% indicating higher maintenance costs, yet no changes were detected in the respiratory capacity of isolated mitochondria. ATO exposure led to ˜60% decrease in the lysosomal membrane stability of hemocytes and ˜3-fold decrease in the whole-organism P-glycoprotein-driven and diffusional efflux of xenobiotics indicating altered membrane properties. The digestive gland was a major target of ATO toxicity in the mussels. Exposure of mussels to ATO led to depletion of lipid, carbohydrate and protein pools, and suppressed transcription of key enzymes involved in mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-alpha PGC-1α) and fatty acid metabolism (acetyl-CoA carboxylase and CYP4Y1) in the digestive gland. No bioenergetic disturbances were observed in the gills of ATO-exposed mussels, and elevated GST activity indicated enhanced ATO detoxification in this tissue. These data demonstrate that ATO can act as a metabolic disruptor and chemosensitizer in keystone marine bivalves and warrant further investigations of statins as emerging pollutants of concern in coastal marine ecosystems.

Interactive effects of osmotic stress and burrowing activity on protein metabolism and muscle capacity in the soft shell clam Mya arenaria.

Bioturbators such as sediment-dwelling marine bivalves are ecosystem engineers that enhance sediment-water exchange and benthic-pelagic coupling. In shallow coastal areas, bivalves are exposed to frequent disturbance and salinity stress that might negatively affect their activity and physiological performance; however, the mechanisms underlying these effects are not fully understood. We investigated the effects of osmotic stress (low and fluctuating salinity) and repeated burrowing on aerobic and contractile capacity of the foot muscle (assessed by the activity of succinate dehydrogenase and myosin ATPase) as well as the levels of organic osmolytes (free amino acids) and biochemical markers of protein synthesis and proteolysis in key osmoregulatory and energy storing tissues (gills and hepatopancreas, respectively) in a common bioturbator, the soft shell clam Mya arenaria. Osmotic stress and exhaustive exercise altered the foot muscle capacity of soft shell clams and had a strong impact on protein and amino acid homeostasis in tissues not directly involved in locomotion. Acclimation to constant low salinity (5 practical salinity units) depleted the whole-body free amino acid pool and affected protein synthesis but not protein breakdown in the gill. In contrast, fluctuating (5-15) salinity increased protein breakdown rate, suppressed protein synthesis, caused oxidative damage to proteins in the gill and selectively depleted whole-body glycine pool. Clams acclimated to normal salinity (15) increased the aerobic capacity of the foot muscle upon repeated burrowing, whereas acclimation to low and fluctuating salinity reduced this adaptive muscle plasticity. Under the normal and low salinity conditions, exhaustive exercise induced protein conservation pathways (indicated by suppression of protein synthesis and catabolism), but this effect was disrupted by fluctuating salinity. These findings indicate that exhaustive exercise and osmotic stress interactively affect whole-body protein homeostasis and functional capacity of the foot muscle in soft shell clams which might contribute to reduced burrowing activity of bivalve bioturbators in osmotically challenging environments such as estuaries and shallow coastal zones.

Effects of mechanical disturbance and salinity stress on bioenergetics and burrowing behavior of the soft-shell clam Mya arenaria.

Bioturbation of sediments by burrowing organisms plays a key role in the functioning of coastal ecosystems. Burrowing is considered an energetically expensive activity, yet the energy costs of burrowing and the potential impacts of multiple stressors (such as salinity stress and wave action) on bioenergetics and burrowing performance of marine bioturbators are not well understood. We investigated the effects of mechanical disturbance and salinity stress on the burrowing behavior, aerobic capacity and energy expense of digging in a common marine bioturbator, the soft-shell clam Mya arenaria from the Baltic Sea (control salinity 15). Mya arenaria showed large individual variability in the burrowing efficiency, with an average of ∼7% of the body energy reserves used per burial. Clams with higher mitochondrial capacity and lower energy expenditure per burial showed higher endurance. Acclimation for 3-4 weeks to low (5) or fluctuating (5-15) salinity reduced the burrowing speed and the number of times the clams can rebury but did not affect the mitochondrial capacity of the whole body or the gill. Acclimation to the fluctuating salinity shifted the predominant fuel use for burrowing from proteins to lipids. Our data indicate that the reduced burrowing performance of clams under the salinity stress is not due to the limitations of energy availability or aerobic capacity but must involve other mechanisms (such as impaired muscle performance). The reduction in the burrowing capacity of clams due to salinity stress may have important implications for survival, activity and ecological functions of the clams in shallow coastal ecosystems.

Effects of pH and bicarbonate on mitochondrial functions of marine bivalves.

Estuarine organisms including mollusks are exposed to periodic oxygen deficiency (hypoxia) that leads to a decrease in intracellular pH and accumulation of bicarbonate (HCO3(-)). These changes can affect cellular bioenergetics; however, their effects on mitochondria of estuarine mollusks are not well understood. We determined the interactive effects of bicarbonate (0-10mM) and pH (7.2 and 6.5) on mitochondrial oxygen consumption (MO2), membrane potential (Δψ) and production of reactive oxygen species (ROS) in two common estuarine bivalves - hard clams Mercenaria mercenaria, and bay scallops Argopecten irradians. In both species, elevated HCO3(-) levels suppressed ADP-stimulated (state 3) MO2 but had little effect on the resting (state 4) respiration. These effects were not mediated by the soluble adenylyl cyclase or cyclic AMP. Effects of the low pH (6.5) on mitochondrial traits were species-specific and depended on the substrate oxidized by the mitochondria. Mild acidosis (pH6.5) had minimal effects on MO2 and Δψ of the bivalve mitochondria oxidizing pyruvate but led to increased rates of ROS production in clams (ROS production could not be measured in scallops). In succinate-respiring mitochondria of clams, mild acidosis suppressed MO2 and increased mitochondrial coupling, while in scallop mitochondria the effects of low pH were opposite. Suppression of mitochondrial oxidative phosphorylation by bicarbonate and/or acidosis may contribute to the metabolic rate depression during shell closure or environmental hypoxia/hypercapnia. These findings have implications for understanding the physiological mechanisms involved in regulation of mitochondrial bioenergetics during hypoxia exposure in estuarine bivalves.

DNA recognition by the Salmonella enterica serovar Typhimurium transcription factor SlyA

The Salmonella regulatory protein SlyA is implicated in virulence, survival in macrophages and resistance to oxidative stress and anti-microbial peptides. SlyA is a member of the MarR family of winged-helix transcription factors. Systematic mutational analysis of the SlyA operator sequence and of the predicted DNA-binding region of SlyA shows that no single base pair in the palindromic SlyA operator sequence is essential for DNA binding, and identifies amino acid residues required to allow SlyA to recognise DNA. Combining the structure-function studies described here and elsewhere with the structures of MarR family proteins suggests a possible model for regulation of SlyA binding to DNA (AU)

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DNA recognition by the Salmonella enterica serovar Typhimurium transcription factor SlyA.

The Salmonella regulatory protein SlyA is implicated in virulence, survival in macrophages and resistance to oxidative stress and anti-microbial peptides. SlyA is a member of the MarR family of winged-helix transcription factors. Systematic mutational analysis of the SlyA operator sequence and of the predicted DNA-binding region of SlyA shows that no single base pair in the palindromic SlyA operator sequence is essential for DNA binding, and identifies amino acid residues required to allow SlyA to recognise DNA. Combining the structure-function studies described here and elsewhere with the structures of MarR family proteins suggests a possible model for regulation of SlyA binding to DNA.

Alternate SlyA and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12.

Haemolysin E is a cytolytic pore-forming toxin found in several Escherichia coli and Salmonella enterica strains. Expression of hlyE is repressed by the global regulator H-NS (histone-like nucleoid structuring protein), but can be activated by the regulator SlyA. Expression of a chromosomal hlyE-lacZ fusion in an E. coli slyA mutant was reduced to 60% of the wild-type level confirming a positive role for SlyA. DNase I footprint analysis revealed the presence of two separate SlyA binding sites, one located upstream, the other downstream of the hlyE transcriptional start site. These sites overlap AT-rich H-NS binding sites. Footprint and gel shift data showed that whereas H-NS prevented binding of RNA polymerase (RNAP) at the hlyE promoter (PhlyE), SlyA allowed binding of RNAP, but inhibited binding of H-NS. Accordingly, in vitro transcription analyses showed that addition of SlyA protein relieved H-NS-mediated repression of hlyE. Based on these observations a model for SlyA/H-NS regulation of hlyE expression is proposed in which the relative concentrations of SlyA and H-NS govern the nature of the nucleoprotein complexes formed at PhlyE. When H-NS is dominant RNAP binding is inhibited and hlyE expression is silenced; when SlyA is dominant H-NS binding is inhibited allowing RNAP access to the promoter facilitating hlyE transcription.