Membrane Of Gram-negative Bacteria Research Articles

There is no association between serum endotoxin levels and inflammation in asthma

Backround Endotoxin, in lipopolysaccharide structure (LPS), is the main component of the outer membrane of gram negative bacteria. LPS levels were associated with inflammatory disease. Asthma is a chronic inflammatory disease involving many different cell types and cellular elements. The association between LPS serum levels and the asthma is not well known. The aim of this study was to investigate the association between the LPS serum levels and the severity of asthma, demographic data and laboratory parameters. Methodology The study included 67 patients aged >18 years with a diagnosis of asthma, and 15 healthy volunteers with no history of chronic disease as a control group. The Asthma Control Test (ACT), Respiratory Function Tests (RFTs), fractional exhaled nitric oxide (FeNO), and endotoxin levels were measured and compared between the groups. The endotoxin measurements were performed using the ELISA method. Results The mild-moderate asthma group included 33 patients and the severe asthma group, 34 patients. The endotoxin level was measured as 17.78 (range 3.59 to 304.55) EU/ml in the patient group and 15 (range 4.01 to 74.06) EU/ml in the control group with no statistically significant difference determined between the groups. In the subgroups, the endotoxin level was measured as 15.21 (range 3.69 to 304.55) EU/ml in the mild-moderate group and 14.46 (range 3.59 to 278.86) EU/ml in the severe asthma group with no statistically significant difference determined between the groups. Conclusion The results of this study showed no relationship between serum endotoxin level and asthma or asthma severity.

Modulation of OMV Production by the Lysis Module of the DLP12 Defective Prophage of Escherichia coli K12.

Outer membrane vesicles (OMVs) are nanostructures mostly produced by blebbing of the outer membrane in Gram negative bacteria. They contain biologically active proteins and perform a variety of processes. OMV production is also a typical response to events inducing stress in the bacterial envelope. In these cases, hypervesiculation is regarded as a strategy to avoid the dangerous accumulation of undesired products within the periplasm. Several housekeeping genes influence the biogenesis of OMVs, including those correlated with peptidoglycan and cell wall dynamics. In this work, we have investigated the relationship between OMV production and the lysis module of the E. coli DLP12 cryptic prophage. This module is an operon encoding a holin, an endolysin and two spannins, and is known to be involved in cell wall maintenance. We find that deleting the lysis module increases OMV production, suggesting that during evolution this operon has been domesticated to regulate vesiculation, likely through the elimination of non-recyclable peptidoglycan fragments. We also show that the expression of the lysis module is negatively regulated by environmental stress stimuli as high osmolarity, low pH and low temperature. Our data further highlight how defective prophages finely contribute to bacterial host fitness.

Characterization and Analysis of Leukotoxin-containing Outer Membrane Vesicles

Outer membrane vesicles (OMVs) are nanoscale, membrane-bound structures that bleb off from the outer membrane of Gram negative bacteria. Although being discovered over 30 years ago, only in the last decade have OMVs garnered attention for their bacterial function. Specifically, they have been shown to be enriched in bacterial toxins as well as other virulence factors, but not all OMVs in a single population will have the same content. Heterogeneity amongst OMV populations, in terms of size, function and composition, has served as a point of debate within the field, resulting in the inability to reach a consensus on important OMV mechanisms. Heterogeneity has often been rejected due to limited analyses. In this work, we have captured leukotoxin-containing OMVs, produced by Aggregatibacter actinomycetemcomitans (Aa), through modifying a quartz crystal microbalance with dissipation monitoring (QCM-D) sensor with an anti-leukotoxin antibody. Our previous work demonstrated that there are two distinct populations of Aa OMVs, one smaller (<100 nm) and one larger (∼300-350 nm) and that leukotoxin is preferentially sorted to the larger OMVs. To further examine this concept, a polymer array with conjugated anti-leukotoxin antibody was assembled on a QCM-D sensor. OMVs were injected over the antibody-functionalized sensor, exclusively capturing the larger OMVs. Fluorescence imaging of individual OMVs also confirms this size heterogeneity based on toxin content. This application provides a biosensing strategy to differentiate OMV subpopulations, especially with respect to the distribution of toxin content as a function of OMV size.

Immunotropic and Immunogenic Properties of Nonspecific Yersinia Porins

The purpose of the study is to summarize our own data and literature data on the signifi cance of poreforming proteins of the outer membrane of Yersinia as factors of their pathogenicity and as diagnostic and protective antigens, and their role in pathological processes considered non-infectious. In the last decades of the last century, the epidemic signifi cance of intestinal yersiniosis caused by the bacteria of Yersinia genus, Yersinia pseudotuberculosis and Yersinia enterocolitica, which are the «doubles» of the plague pathogen (Yersinia pestis) by genetic, cultural, biochemical, and other properties, has signifi cantly increased. It has been established that acute yersiniosis infections without eff ective treatment can pass into secondary focal forms, leading to the development of systemic diseases that were not previously considered infectious (for example, Grave’s disease). Th ey are characterized by multiple organ lesions, dysfunctions of the cardiovascular and nervous systems, musculoskeletal system, urinary system and gastrointestinal tract, which is a consequence of autoimmune processes based on the ability of Yersinia to molecular mimicry. Th e paper shows that porins play a signifi cant role in the development of the infectious process and can be considered as pathogenic factors of bacteria. Together with other components of the outer membrane of gram negative bacteria, they provide adhesion, invasion and colonization of the cells of the host organism by bacteria. Porins can aff ect a number of eukaryotic cell functions, including cytokine expression, receptor activation, apoptosis induction, and regulation of the actin cytoskeleton. It has been established that pore-forming Yersinia proteins are protective antigens. The administration of them to laboratory animals induces the formation of species-specifi c immunity, which allows us to recommend porin protein as a component of chemical and genetic engineering vaccines. Porins are promising for the development of ELISA test systems for the diagnosis of pseudotuberculosis and intestinal yersiniosis, as well as immunopathologies caused by Yersinia bacteria. Given the high degree of similarity of the primary structures of Yersinia porins, it can be assumed that protective preparations created on their basis will protect against infections caused by intestinal yersiniosis, pseudotuberculosis and plague pathogens

Functional Relevance of Unstructured Regions of AcrA, the Periplasmic Adaptor of the Major Multidrug Efflux System in E. coli

Efflux pumps, combined with low permeability of the outer membrane of gram negative bacteria play a crucial role in bacteria multi‐drug resistance. There are five classes of efflux pumps but the major and most studied of the efflux system in E. coli is the resistance nodulation cell division (RND) pump. This pump is a complex of three proteins: the inner membrane transporter AcrB, the periplasmic adaptor AcrA, and the outer membrane protein channel TolC. Of the three proteins, the structure and dynamic behavior of AcrA remain to be fully elucidated. In this study the functional roles of the unstructured regions at the N‐ and C‐termini of the protein were examined. These structurally unresolved regions include residues 26–37 at the N‐terminus, and residues 376 to 397 at the C‐terminus. We conducted truncation mutation to systematically remove residues from the C‐terminus of AcrA, and found that residues after Q376 are indeed not functionally important. The truncated AcrA, in which E377 is replaced by a stop codon, is fully active. On the N‐terminus, we created a mutant in which four amino acid residues (Q31QGG34) were deleted. The mutant was nonfunctional. To understand the mechanism of function disruption, we modified the membrane anchoring of AcrA, and determined the role of these first 13 residues at the N‐terminus of the mature AcrA sequence.Support or Funding InformationNSF grant CHE‐1709381This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Polychlorinated Pesticides and Their Metabolites on Phospholipid Organization in Model Microbial Membranes.

Polychlorinated pesticides (PPs) were classified as persistent organic pollutants because of their toxicity, limited degradability in the environment, bioaugmentation, and accumulation in animal tissues. PPs accumulate in the environment mainly in the soils and water sediments where they are toxic to the decomposer organisms including soil bacteria and fungi. Therefore, there is an urgent need to search for the microorganisms capable of PP biodegradation which could be applied for soil bioremediation. The exact mechanism of PP microbial toxicity is unknown; however, there is evidence that it can be membrane related. To shed light on the interactions of PPs with microbial membranes, we applied Langmuir monolayers formed by phospholipids as model biomembranes. The model membranes were formed by phospholipids typical to microbial membranes: cardiolipins and phosphatidylglycerols the main components of Gram positive bacteria membranes, phosphatidylcholine typical to fungal membranes, as well as phosphatidylethanolamine found in the inner membranes of Gram negative bacteria. For the studies, the most ecotoxic PPs and their water-soluble metabolites were chosen. The monolayers were studied with the application of mutually complementary techniques: Langmuir technique, grazing incidence X-ray diffraction, and PM-IRRAS spectroscopy. It turned out that the cyclodiene PPs are more membrane active than monocyclic PPs and that the possibility of their incorporation is strictly related to the phospholipid structure. The membranes prepared with cardiolipin turned out to be especially resistant to PP incorporation. Regarding the metabolites, pentachlorophenol turned out to be especially structure breaking, affecting the molecular organization of all of the investigated phospholipids.

Building Blocks of the Outer Membrane: Calculating a General Elastic Energy Model for β-Barrel Membrane Proteins.

The outer membranes of Gram negative bacteria are the first points of contact these organisms make with their environment. Understanding how composition determines the mechanical properties of this essential barrier is of paramount importance. Therefore, we developed a new computational method to measure the elasticity of transmembrane proteins found in the outer membrane. Using all-atom molecular dynamics simulations of these proteins, we apply a set of external forces to mechanically stress the transmembrane β-barrels. Our results from four representative β-barrels show that outer membrane proteins display elastic properties that are approximately 70 to 190 times stiffer than neat lipid membranes. These findings suggest that outer membrane β-barrels are a significant source of mechanical stability in bacteria. Our all-atom approach further reveals that resistance to radial stress is encoded by a general mechanism that includes stretching of backbone hydrogen bonds and tilting of β-strands with respect to the bilayer normal. This computational framework facilitates an increased theoretical understanding of how varying lipid and protein amounts affect the mechanical properties of the bacterial outer membrane.

Imperatorin ameliorates lipopolysaccharide induced memory deficit by mitigating proinflammatory cytokines, oxidative stress and modulating brain-derived neurotropic factor

BackgroundLipopolysaccharide (LPS), an endotoxin from the outer membrane of Gram negative bacteria has been reported to cause neuroinflammation and learning and memory deficits. There are reports describing the beneficial effects of Imperatorin (IMP), a naturally occurring furanocoumarin in central nervous system (CNS) disorders such as anxiety and epilepsy. ObjectiveIn the current study, we investigated whether IMP protects against LPS mediated memory deficits and neuroinflammation. MethodsMice pretreated with IMP (5, 10 mg/kg po) were administered LPS (250 μg/kg ip) for 7 days. Memory was evaluated in the Morris water maze (MWM) and Y maze. The mice were euthanised and different biochemical assessments were carried out to measure oxidative stress and acetyl choline esterase (AChE). Further, evaluation of proinflammatory cytokines such as tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) levels and brain derived neurotrophic factor (BDNF) in hippocampus and cortex of brain were performed. ResultsLPS administration caused poor memory retention in both, MWM and Y maze, and caused distinct oxidative stress since decrease in superoxide dismutase (SOD), reduced glutathione (GSH) levels and increased lipid peroxidation were observed. Also, a significant rise was observed in the levels of AChE. Moreover, a rise in TNF-α and IL-6 levels and depleted levels of BDNF were noted. IMP pretreatment reversed LPS induced behavioral and memory disturbances and significantly decreased the oxidative stress and AChE levels. It also reduced TNF-α and IL-6 levels and caused a significant upregulation of BDNF levels. ConclusionPresent study highlights the potential neuroprotective role of IMP against LPS mediated cognitive impairment and neuroinflammation.

Lipopolysaccharide Prompts Oxidative Stress and Apoptosis in Rats’ Testicular Tissue

Lipopolysaccharide (LPS) is a component of the outer membrane of gram negative bacteria. LPS challenging allows switching transcription of proinflammatory cytokines on via over stimulation of Toll-like receptors (TLRs) signaling pathway with subsequent pathogenic inflammatory response. We investigated the possible reproductive toxicity of LPS in male Wister albino rats. Oxidative stress markers, antioxidant status and caspase-3 activity were analyzed in testicular tissues of rats exposed to either saline or LPS (4 mg/kg BW, ip; 0.18 of the LD50). The samples were collected at 6 h and 72 h after injection of LPS. A significant reduction in testicular reduced glutathione (GSH), glutathione-S-transferase (GST) and superoxide dismutase (SOD) was observed at 72 h compared to control group. Total antioxidant capacity was decreased at 6 h with additional significant reduction at 72 h. Catalase activity was reduced significantly at both 6 and 72 h. Malondialdehyde (MDA) was increased (P ≤ 0.05) in LPS injected rats without variation between 6 and 72 h. A significant increase in nitric oxide (NO) was observed at 72 h after injection. A time-dependent increase in LPS-treated groups was observed in the concentration of caspase-3.Histopathological analysis revealed degenerative changes and necrosis of seminiferous tubules after 6 h with further accumulation of eosinophilic edematous transudate in its lumen after 72 h. In conclusion, by increasing time of exposure, LPS induced lipid peroxidation, oxidative stress, reduced testicular antioxidant capacity and encouraged testicular apoptosis which could be possible mechanisms for impairment of testicular function.

A tethered bilayer lipid membrane that mimics microbial membranes.

A model membrane system has been developed, which mimics the outer membrane of Gram negative bacteria. The structure is based on a tethered monolayer which has been fused with vesicles containing lipopolysaccharide molecules. The effect of the composition of the monolayer and the lipids in the outer layer on the structural and electrical properties of the membrane has been investigated. By using electrochemical impedance spectroscopy as well as neutron scattering techniques, it could be shown that a relatively high tethering density and a small amount of diluting lipids in the outer membrane leaflet leads to the formation of a stable solid supported membrane. The influence of divalent ions on the membrane stability has been probed as well as the interaction of the bilayer with the antibiotic colistin. A number of different architectures were developed, suited to both the study of bacterial membrane proteins and the screening of antimicrobial activity of potential drug candidates.

Lipopeptide-induced changes in permeability of solid supported bilayers composed of bacterial membrane lipids

Lipopeptides are known to show bactericidal activity and due to their simple structure, ease of design, and low cost of implementation, they are often considered potent replacement for many traditional antibiotics. Another important advantage of lipopeptides is related to the fact that their preferential target is a cell membrane. Hence their action is less specific than conventional antibiotics, which means that development of drug resistance by pathogens is less probable in such case. In this paper we have utilized electrochemical methods and in situ atomic force microscopy to evaluate the mode of action of novel lipopeptide C15H31CO-DPhe-Dab-Dab-Leu-NH2 on planar lipid bilayer. The latter was composed of phosphatidylethanolamines and phosphatidylglycerols extracts from E. coli. Therefore it can be considered as a simplified model of inner membrane of Gram negative bacteria. We have found that lipopeptide-lipid interactions strongly affect molecular organization of PE/PG bilayer, which is reflected by increased disorder and subsequent perforation of the film. Importantly, fluid domains were identified as preferential sites for insertion of lipopeptide molecules, which tend to accumulate within the membrane. However, above certain threshold ratio the membrane becomes swollen and strongly destabilized. This results in membrane rupture and large mixed lipopeptide-lipid aggregates departure from electrode surface. Based on experimental observations, the mechanism of C16-fXXL action on bacterial-like model membrane is proposed.

Detection of endotoxins using nanomaterials

An endotoxin is a lipopolysaccharide complex that is associated with the outer membrane of gram negative bacteria, and even a small amount can trigger strong exothermic activity. Pharmaceutical and food products can be contaminated with endotoxins, so there is a strict need to control them. A limulus amoebocyte lysate (LAL) assay is a standard endotoxin detection method that is highly sensitive and easy to use, but it has disadvantages in that the sample preparation is a complex process, it takes a long time to confirm the presence of endotoxins, and it requires a controlled workplace. Recently, endotoxin detection using nanomaterials has been gaining attention, and this review reports on the latest endotoxin detection methods using gold nanoparticles, magnetic nanoparticles, quantum dots, and carbon materials, among different nanomaterials, and it also introduces the future prospect of endotoxin detection.

Sialic acid acquisition in bacteria-one substrate, many transporters.

The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, β-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell.

Neutrophil Infiltration Is Partially Inhibited By EC-18 in the LPS-Induced Acute Lung Injury

Acute lung injury (ALI) is an acute respiratory failure and linked closely to neutrophil accumulation. It can lead to acute respiratory distress syndrome (ARDS). Mouse model of ALI was established by lipopolysaccharide (LPS) administration. LPS, an outer membrane of gram negative bacteria, is considered as immune stimulator via recognition as pathogen-associated molecular patterns (PAMP). ALI and neutrophil infiltration were readily induced by intranasal injection of LPS. In this study, we investigated whether EC-18 treatment attenuates LPS-induced ALI. EC-18 (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is an immune modulator in the allergic asthma response through modulation of the balance between Th1 and Th2. To analyze the role of EC-18 in LPS-induced ALI mice, Balb/c mice were divided into three separate groups: control, LPS treated, and EC-18/LPS co-treated (n=5 per group). 25 mg/kg of LPS was administered by an intranasal route, and 250 mg/kg of EC-18 was orally administered. Mice were sacrificed after 15hrs, and various samples were collected. Bone marrow cells, whole blood cells, cells in lung and bronchoalveolar lavage fluid (BALF) were analyzed using complete blood count (CBC) assay. As results, the number of neutrophil in the bone marrow was decreased in the LPS treated group, and the circulating neutrophil, neutrophil in the lung and BALF were significantly increased in the LPS treated group. Neutrophils in the lung and BALF were dramatically decreased in the EC-18/LPS co-treated group. Also, Evans Blue staining of the lung indicated that capillary permeability was enhanced in the LPS injected mice, and this permeability was lessened in the EC-18/LPS co-treated group as much as that of control. These findings suggested that EC-18 could effectively block neutrophil transmigration into the lung and vesicular leakage. Consequently, EC-18 could be utilized as a potential therapeutic agent for acute and chronic inflammation related disease like ALI. DisclosuresNo relevant conflicts of interest to declare.

The antimicrobial peptide microcin J25 stabilizes the gel phase of bacterial model membranes

The bacterial membrane interaction of the antimicrobial peptide microcin J25 was studied with the probe-free techniques Langmuir monolayers and infrared spectroscopy. Membrane model systems composed by phosphatidylethanolamine:phosphatidylglycerol 7:3, which mimic the cytoplasmic membrane of Gram negative bacteria, were used in both monolayer and bilayer approaches. The peptide reduced the transition surface pressure of the expanded-to-condensed lipid monolayer states, as well as increased the gel-to-liquid crystalline transition temperature in bilayers, indicating a stabilization of membrane ordered state. In addition, a reduction of the surface pressure at which condensed domains appeared was observed upon mixed monolayers compression after microcin J25 adsorption. The results indicate a favorable interaction of microcin J25 with bacterial membrane model systems. Also, the effects on the ordered phases stabilization are discussed in terms of the biological effects observed in membranes of sensitive cells.

Sequence context induced antimicrobial activity: insight into lipopolysaccharide permeabilization

Lactoferrampin (WR17, Trp 268-Arg 284), an antimicrobial peptide, is known to have significant antibacterial and candidacidal activities. However, there are no previous studies explaining how WR17 permeabilizes the outer membrane of Gram negative bacteria and neutralizes endotoxins. In this study we used a series of assays like antimicrobial activity, calcein leakage, NPN dye uptake and endotoxin neutralization assay to show that the sequence context of WR17 modulates its multi-faceted activities. We determined the high resolution NMR structure of WR17 in LPS and found that the N-ter region forms a helix (Trp1-Phe11) and orients itself at an angle of 45° into the lipopolysaccharide (LPS) micelle, whereas the C-ter region (Lys13-Arg17) remains as a flexible extended random coil. We also verified this result through in silico molecular modeling simulation. Isothermal titration calorimetry showed that the interaction of WR17 and its analogues with LPS was primarily endothermic in nature. Using several fluorescence techniques such as anisotropy and red edge excitation shift assay we revealed motional restriction for Trp1 of WR17 in LPS. The distance between the indole ring of Trp1 of WR17 and the polar head group of LPS is around 7 Å, as obtained from the depth of insertion assay. Additionally, MD simulation demonstrated that the incorporation of the peptide in LPS is achieved with the help of the K(13)xK(15)xR(17) motif at the C-terminus. This novel anchoring "K(13)NKSR(17)" motif is currently being utilized in our ongoing research to design novel anti-endotoxic molecules.

Effect of Colostrum on the Symptoms and Mucosal Permeability in Patients with Irritable Bowel Syndrome: A Randomized Placebo-controlled Study.

Effect of Colostrum on the Symptoms and Mucosal Permeability in Patients with Irritable Bowel Syndrome: A Randomized Placebo-controlled Study.

NMR Structure of Temporin-1 Ta in Lipopolysaccharide Micelles: Mechanistic Insight into Inactivation by Outer Membrane

BackgroundAntimicrobial peptides (AMPs) play important roles in the innate defense mechanism. The broad spectrum of activity of AMPs requires an efficient permeabilization of the bacterial outer and inner membranes. The outer leaflet of the outer membrane of Gram negative bacteria is made of a specialized lipid called lipopolysaccharide (LPS). The LPS layer is an efficient permeability barrier against anti-bacterial agents including AMPs. As a mode of protection, LPS can induce self associations of AMPs rendering them inactive. Temporins are a group of short-sized AMPs isolated from frog skin, and many of them are inactive against Gram negative bacteria as a result of their self-association in the LPS-outer membrane.Principal FindingsUsing NMR spectroscopy, we have determined atomic resolution structure and characterized localization of temporin-1Ta or TA (FLPLIGRVLSGIL-amide) in LPS micelles. In LPS micelles, TA adopts helical conformation for residues L4-I12, while residues F1-L3 are found to be in extended conformations. The aromatic sidechain of residue F1 is involved in extensive packing interactions with the sidechains of residues P3, L4 and I5. Interestingly, a number of long-range NOE contacts have been detected between the N-terminal residues F1, P3 with the C-terminal residues S10, I12, L13 of TA in LPS micelles. Saturation transfer difference (STD) NMR studies demonstrate close proximity of residues including F1, L2, P3, R7, S10 and L13 with the LPS micelles. Notably, the LPS bound structure of TA shows differences with the structures of TA determined in DPC and SDS detergent micelles.SignificanceWe propose that TA, in LPS lipids, forms helical oligomeric structures employing N- and C-termini residues. Such oligomeric structures may not be translocated across the outer membrane; resulting in the inactivation of the AMP. Importantly, the results of our studies will be useful for the development of antimicrobial agents with a broader spectrum of activity.

Biochemical pathways of amino acids in soil: Assessment by position-specific labeling and 13C-PLFA analysis

Microbial utilization is a key transformation process of soil organic matter (SOM). For the first time, position-specific 13C labeling was combined with compound-specific 13C-PLFA analysis to trace metabolites of two amino acids in microbial groups and to reconstruct detailed biochemical pathways. Short-term transformation was assessed by applying position-specifically 13C labeled alanine and glutamic acid to soil in a field experiment. Microbial utilization of the amino acids' functional groups was quantified by 13C incorporation in total microbial biomass and in distinct microbial groups classified by 13C-PLFA.Loss from PLFAs was fastest for the highly oxidized carboxyl group of both amino acids, whereas the reduced C positions, e.g. C3–5, were preferentially incorporated into microorganisms and their PLFAs. The incorporation of C from alanines' C2 position into the cell membrane of gram negative bacteria was higher by more than one order of magnitude than into all other microbial groups. Whereas C2 of alanine was still bound to C3 at day 3, the C2 and C3 positions were partially split at day 10. In contrast, the C2 of glutamic acid was lost faster from PLFAs of all microbial groups. The divergence index, which reflects relative incorporation of one position to the incorporation of C from all positions in a molecule, revealed that discrimination between positions is highest in the initial reactions and decreases with time.Reconstruction of microbial transformation pathways showed that the C2 position of alanine is lost faster than its C3 position regardless of whether the molecule is used ana- or catabolically. Glutamic acid C2 is incorporated into PLFAs only by two out of eight microbial groups (fungi and part of gram positive prokaryotes). Its incorporation in PLFA can only be explained by either the utilization of the glyoxolate bypass or the transformation of glutamic acid into aspartate prior to being fed into the citric acid cycle. During these pathways, no C is lost as CO2 but neither is energy produced, making them typical C deficiency pathways. Glutamic acid is therefore a promising metabolic tracer in regard to ecophysiology of cells and therefore changing environmental conditions.Analyzing the fate of individual C atoms by position-specific labeling allows insight into the mechanisms and kinetics of microbial utilization by various microbial groups. This approach will strongly improve our understanding of soil C fluxes.

Physical interactions of fish protamine and antisepsis peptide drugs with bacterial membranes revealed by combination of specular x-ray reflectivity and grazing-incidence x-ray fluorescence

As a defined model of outer membranes of gram negative bacteria, we investigated the interaction of monolayers of lipopolysacchrides from Salmonella enterica rough strains R90 (LPS Ra) with natural and synthetic peptides. The fine structures perpendicular to the membrane plane and the ion distribution near the interface were determined by specular x-ray reflectivity (XRR) and grazing-incidence x-ray fluorescence (GIXF) in the presence and absence of divalent cations. The unique combination of XRR and GIXF allows for the quantitative identification of different modes of interactions in a high spatial resolution, which cannot be assessed by other experimental methods. Natural fish protamine disrupts the stratified membrane structures in the absence of Ca(2+) ions, while staying away from the membrane surface in the presence of Ca(2+) ions. In contrast, synthetic antisepsis peptide Pep 19-2.5 weakly adsorbs to the membrane and stays near the uncharged sugar units even in the absence of Ca(2+). In the presence of Ca(2+), Pep 19-2.5 can reach the negatively charged inner core without destroying the barrier capability against ions.