Morphological analysis of young and old pellicles of Salmonella Typhimurium

Abstract

A wide variety of microorganisms are able to form biofilms at the interface between air and liquid (pellicles). In this study changes during the maturation of the pellicle of Salmonella Typhimurium were analysed and the role of cellulose in the pellicle structure and morphology evaluated. The morphology of both sides of the pellicle was characterised using atomic force microscopy and scanning electron microscopy. Overall, there was a marked difference in the morphology of the water-facing (WF) and air-facing (AF) biofilm surfaces. While the AF side appeared to be uniform, and extensively covered with an exocellular coating, cells in the WF side were distributed into clusters and were less covered. However, the similarity in size and shape of single cells from both sides of the pellicle may indicate that the bacterial cells across the pellicle have a similar physiological status. During maturation, porous structures with multiple cracks and channels were created in the pellicle, leading to disintegration. By comparison with the structure of pellicles of a cellulose-deficient mutant, it was demonstrated that the observed disintegration of mature pellicles probably occurred in part by self-hydrolysis of components of the matrix.     

Thylakoid membrane remodeling during state transitions in Arabidopsis

Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.     

High eradication rates of Helicobacter pylori infection following sequential therapy: the Israeli experience treating naïve patients

Background: Helicobacter pylori eradication rates following triple therapy are decreasing. Cure rates as low as 57%, mainly to claritromycin resistance, have been reported in Israel. Studies performed in Italy have shown eradication rates of 93%, following sequential therapy. Our aim was to evaluate the effect of sequential therapy on eradication rates of H. pylori in naïve Israeli patients. Material and Methods: Consecutive patients referred for esophagogastroduodenoscopy with a positive rapid urease test and positive ¹³C urea breath test were included. Patients received omeprazole 20 mg bid and amoxicillin 1 g bid for 5 days followed by omeprazole 20 mg bid, clarithromycin 500 mg bid and tinidazole 500 mg bid for the subsequent 5 days. A second ¹³C urea breath test was performed at least 4 weeks after completion of therapy. Patients were asked to avoid antibiotics, bismuth compounds or proton pump inhibitor until after the second ¹³C urea breath test. Adverse effects were documented by a questionnaire. Results: One hundred and twenty‐four patients (mean age 56.1 ± 12.5 years, 55.6% women) were included; 120/124 (96.8%) completed treatment and performed the second ¹³C urea breath test. Two patients (1.6%) were lost to follow‐up; 2 (1.6%) were noncompliant with study regulations. One hundred and fifteen patients achieved eradication of H. pylori. The eradication rate was 95.8% by per protocol analysis and 92.7% by intention to treat analysis. Conclusion: The sequential regimen attained significantly higher eradication rates in naïve patients than usually reported for conventional triple therapy. Sequential therapy may be an alternative first‐line therapy in eradicating H. pylori in Israel.     

The Association of Peroxidase Activity and Resistance of Maize to Exserohilum turcicum

Peroxidase activity in leaves of maize (Zea mays L.) differing in susceptibility to Exserohilum turcicum has been investigated in relation to their resistance to Northern Leaf Blight (NLB) caused by the fungal pathogen E. turcicum. In non-inoculated plants, high peroxidase activity was detected in leaves of the resistant isolines B37HtN and B73HtN as compared with the susceptible isolines B37 and B73 and the sweet corn variety Jubilee. After inoculation with E. turcicum, peroxidase activity increased in both susceptible and resistant isolines B73 and B73HtN. However, marked enhancement of peroxidase activity was detected 6 days after inoculation and became remarkable in isoline B73HtN, although symptomes started to show up in both susceptible and resistant plants only 10 days after inoculation. Using polyacrylamide gel electrophoresis separations, different banding pattern of isoperoxidases was found in the susceptible plants as compared with the resistant ones. In non-inoculated plants, three differential bands which appeared in the resistant isoline B37HtN, were absent in the susceptible Jubilee plants, and were as traces in the isoline B37. These bands first appeared in Jubilee and as clear bands in B37, only after inoculation with E. turcicum. The association of these isoperoxidases and resistance of maize to E. turcicum is discussed.     

Origin of β-Carotene-Rich Plastoglobuli in Dunaliella bardawil

The halotolerant microalgae Dunaliella bardawil accumulates under nitrogen deprivation two types of lipid droplets: plastoglobuli rich in β-carotene (βC-plastoglobuli) and cytoplasmatic lipid droplets (CLDs). We describe the isolation, composition, and origin of these lipid droplets. Plastoglobuli contain β-carotene, phytoene, and galactolipids missing in CLDs. The two preparations contain different lipid-associated proteins: major lipid droplet protein in CLD and the Prorich carotene globule protein in βC-plastoglobuli. The compositions of triglyceride (TAG) molecular species, total fatty acids, and sn-1+3 and sn-2 positions in the two lipid pools are similar, except for a small increase in palmitic acid in plastoglobuli, suggesting a common origin. The formation of CLD TAG precedes that of βC-plastoglobuli, reaching a maximum after 48 h of nitrogen deprivation and then decreasing. Palmitic acid incorporation kinetics indicated that, at early stages of nitrogen deprivation, CLD TAG is synthesized mostly from newly formed fatty acids, whereas in βC-plastoglobuli, a large part of TAG is produced from fatty acids of preformed membrane lipids. Electron microscopic analyses revealed that CLDs adhere to chloroplast envelope membranes concomitant with appearance of small βC-plastoglobuli within the chloroplast. Based on these results, we propose that CLDs in D. bardawil are produced in the endoplasmatic reticulum, whereas βC-plastoglobuli are made, in part, from hydrolysis of chloroplast membrane lipids and in part, by a continual transfer of TAG or fatty acids derived from CLD.Eukaryotic cells accumulate neutral lipids in different tissues mainly in the form of lipid droplets (Murphy, 2012). Most lipid droplets consist of a core of triglycerides (TAGs) and/or sterol esters coated by a phospholipids monolayer and embedded with proteins (Zweytick et al., 2000). Plants accumulate TAGs in different tissues, primarily in seeds but also in fruit, such as palm oil, flowers, and leaves. The best characterized system for TAG metabolism is oil seeds, in which TAG serves as the major carbon and energy reservoir to be used during germination (Huang, 1992, 1996). Recent studies show that lipid droplets are not just static pools of lipids but have diverse metabolic functions (Farese and Walther, 2009). In addition, plants also contain plastoglobuli, small chloroplastic lipid droplets consisting primarily of storage lipids and pigments. Proteome analyses of plastoglobuli suggest that they are involved in synthesis and degradation of lipids, pigments, and coenzymes (Ytterberg et al., 2006; Lundquist et al., 2012). It has been shown that plant plastoglobuli are associated with thylakoid membranes (Austin et al., 2006; Ytterberg et al., 2006).It is not entirely clear where the TAGs are synthesized in the plant cell. Until recently, it has been assumed that most TAGs are made in the endoplasmatic reticulum (ER) from fatty acids, which are mostly synthesized in the chloroplast and imported to the cytoplasm (Joyard et al., 2010). However, the recent identification of the enzyme diacylglycerol acyl transferase in plant plastoglobuli (Lundquist et al., 2012) suggests that TAG may be synthesized directly in chloroplasts, although direct evidence is missing. TAG may be synthesized also from galactolipid fatty acids during stress or senescence by phytyl ester synthases, which catalyze acyl transesterification from galactolipids to TAGs (Lippold et al., 2012). Phosphatidyl choline (PC) plays a major role in acyl transfer of newly synthesized fatty acids from the chloroplast into TAGs at the ER in plants (Bates et al., 2009). An indication for the origin of glycerolipids in plants is the identity of the fatty acids at the sn-2 position: if it originates in the chloroplast, it is mostly C16:0, whereas if it was made in the ER, it is mostly C:18 (Heinz and Roughan, 1983).Many species of unicellular microalgae can accumulate large amounts of TAGs under growth-limiting conditions, such as nitrogen deprivation (Shifrin and Chisholm, 1981; Roessler, 1990; Avron and Ben-Amotz, 1992; Thompson, 1996). In green microalgae (Chlorophyceae), TAGs are usually synthesized and accumulated in cytoplasmatic lipid droplets (CLDs; Murphy, 2012), although in some cases, such as in Chlamydomonas reinhardtii starchless mutants, they also accumulate in chloroplasts (Fan et al., 2011; Goodson et al., 2011). Recent studies indicate that the CLDs are closely associated with ER membranes and possibly, chloroplast envelope membranes as well (Goodson et al., 2011; Peled et al., 2012).Green microalgae also contain two distinct types of chloroplastic lipid droplets. The first type is plastoglobuli, similar in morphology to higher plants plastoglobuli (Bréhélin et al., 2007; Kessler and Vidi, 2007). The second type is the eyespot (stigma), part of the visual system in microalgae. The eyespot is composed of a cluster of β-carotene-containing lipid droplets organized in several layers between grana membranes in the chloroplast (Häder and Lebert, 2009; Kreimer, 2009). Recent proteomic analysis of algal eyespot proteins revealed that they contain diverse structural proteins, lipid and carotenoid metabolizing enzymes, transporters, and signal transduction components (Schmidt et al., 2006).The origin of TAG in microalgae is still not clear. In C. reinhardtii, it was found that the major fatty acids in the sn-2 position are 16:0, which according to the plant dogma, is made in the chloroplast (Fan et al., 2011). In C. reinhardtii, which lacks PC, monogalactosyldiacylglycerol (MGDG) was proposed to replace PC in the mobilization of fatty acids from plastidal galactoglycerolipids into TAG based on mutation of a galactoglycerolipid lipase (Li et al., 2012). Based on these results and others, it has been proposed that, in C. reinhardtii, triglycerides are primarily produced in the chloroplast or combined with ER (Li et al., 2012; Liu and Benning, 2013).Plants and algae lipid droplets contain structural major proteins localized at the lipid droplet periphery, and their major function seems to be stabilization and prevention of fusion (Huang, 1992, 1996; Katz et al., 1995; Frandsen et al., 2001; Liu et al., 2009). In plant seed oils, the major classes of lipid droplet proteins are oleosins and caleosins, which have a characteristic hydrophobic loop with a conserved three Pro domain (Hsieh and Huang, 2004; Capuano et al., 2007; Purkrtova et al., 2008; Tzen, 2012). Oleosin and caleosin analogs were also recently identified in some green microalgal species (Lin et al., 2012; Vieler et al., 2012; Huang et al., 2013). However, the most abundant lipid droplets proteins in green algae (Chloropyceae) are a new family of major lipid droplet proteins (MLDPs) structurally distinct from plant oleosins and caleosins (Moellering and Benning, 2010; Peled et al., 2011; Davidi et al., 2012). Plastoglobules have different major lipid-associated proteins termed plastoglobules-associated protein-fibrillins, which form a distinct protein family with no sequence or structural similarities to oleosins (Kim and Huang, 2003). We have previously identified in the plastoglobuli rich in β-carotene (βC-plastoglobuli) a lipid-associated protein termed carotene globule protein (CGP), whose degradation destabilized the lipid droplets (Katz et al., 1995). The proteome of C. reinhardtii lipid droplet indicates that algal CLDs also contain several enzymes, suggesting that they are involved in lipid metabolism (Nguyen et al., 2011).The halotolerant green algae Dunaliella bardawil and Dunaliella salina ‘Teodoresco’ are unique in that they accumulate under high light stress or nitrogen deprivation large amounts of plastidic lipid droplets (βC-plastoglobuli), which consist of TAG and two isomers of β-carotene, all trans and 9-cis (Ben-Amotz et al., 1982, 1988). D. bardawil also accumulates CLD under the same stress conditions, similar to other green algae (Davidi et al., 2012). It has been shown that the function of βC-plastoglobuli is to protect the photosynthetic system against photoinhibition (Ben-Amotz et al., 1989). The enzymatic pathway for β-carotene synthesis in D. bardawil and D. salina has been partly identified, but the subcellular localization of β-carotene biosynthesis is not known (Jin and Polle, 2009). The synthesis of β-carotene depends on TAG biosynthesis (Rabbani et al., 1998); however, the origin of βC-plastoglobuli is not known. Are they formed within the chloroplast, or are they made in the cytoplasm? Is the TAG in βC-plastoglobuli and CLD identical or different, and where is it formed?D. bardawil is an excellent model organism for isolation of lipid droplet for several reasons. First, D. bardawil contains large amounts of both CLD and βC-plastoglobuli (Ben-Amotz et al., 1982; Fried et al., 1982), making it possible to obtain sufficient amounts of proteins and lipids from the two types of lipid pools for detailed analyses. Second, Dunaliella do not have a rigid cell wall and can be lysed by a gentle osmotic shock, which does not rupture the chloroplast. Therefore, it is possible to sequentially release pure CLD and βC-plastoglobuli by a two-step lysis (Katz et al., 1995). Third, D. bardawil seems to lack the eyespot structure, which can be clearly observed in other Dunaliella spp. even in a light microscope or by electron microscopy, but has never been observed in D. bardawil by us. It avoids the risk of cross contamination of βC-plastoglobuli with eyespot proteins. Fourth, the availability of protein markers for the major lipid droplet-associated proteins, CGPs and MLDPs, enabled both good immunolocalization and careful monitoring of the purity of the preparations by western analysis.In this work, we describe the purification, lipid compositions, and protein profiles of two lipid pools from D. bardawil: CLD and plastidic βC-plastoglobuli. A detailed proteomic analysis of these lipid droplets will be described in another work. Combined with detailed electron microscopy studies, these results led to surprising conclusions regarding the origin of the plastidic βC-plastoglobuli.     

Utilization of amylose-lipid complexes as molecular nanocapsules for conjugated linoleic Acid

Amylose-conjugated linoleic acid (CLA) complexes were produced by water/dimethyl sulfoxide (DMSO) and KOH/HCl complexation methods. The formation of amylose V form was confirmed by X-ray diffraction (XRD), and complexes formed at 30, 60, and 90 degrees C exhibit melting temperatures exceeding 88 degrees C. Atomic force microscopy (AFM) images showed distinct difference in complex organization, with complexes formed in water/DMSO showing spherical shape with typical diameter of 150 nm. Complexes formed by KOH/HCl showed elongated structure with typical width of 43-160 nm. Water/DMSO complexes exhibit superior protection to CLA against oxidation. All complexes showed high retention of CLA in simulated stomach conditions, and the digestion of complexes by amylases results in high hydrolysis and CLA release by pancreatin and alpha-amylase. Only moderate release was detected following hydrolysis by amyloglucosidase and beta-amylase. It is therefore suggested that amylose-CLA complexes can serve as molecular nanocapsules for protection and delivery of CLA.     

Hypothyroidism Decreases the ATP Sensitivity of KATP Channels from Rat Heart

The effects of thyroid status on the properties of ATP-sensitive potassium channels were investigated. Single-channel recordings were made using excised inside-out membrane patches from enzymatically dissociated ventricular myocytes from hearts of control and thyroidectomized rats and each group was studied with and without administration of thyroid hormone. In patches excised from hypothyroid myocytes the IC₅₀ for ATP inhibition of K_ATP channels was 110 μm. This value was 3-fold higher than the IC₅₀ in control myocytes (43 μm). Treatment of hypothyroid rats to restore physiological levels of thyroid hormone (tri-iodothyronine, T₃), resulted in a return to normal ATP-sensitivity (IC₅₀= 46 μm). In patches from animals rendered hyperthyroid, the IC₅₀ for ATP was 50 μm and this value was not significantly different from the control. There was no difference in the cooperativity of ATP-binding (Hill coefficient, n_H) among control (n_H= 2.2), hypothyroid (n_H= 2.1), T₃-treated (n_H= 2.0) and hyperthyroid groups (n_H= 2.4). The unitary conductance was unchanged and there was no apparent change in intraburst kinetics between examples of single K_ATP channels from control and hypothyroid rats. Action potentials recorded in myocytes from hypothyroid rats were significantly shortened by 50 μm levcromakalim, a K_ATP channel opener (P < 0.001) but unchanged in control myocytes. We conclude that hypothyroidism significantly decreased the ATP-sensitivity of K_ATP channels, whereas the induction of hyperthyroid conditions did not alter the ATP-sensitivity of these channels. Thus, hypothyroidism is likely to have important physiological consequences under circumstances in which K_ATP channels are activated, such as during ischemia. Received: 1 July 1997/Revised: 24 December 1997     

Changes in atmospheric CO2 influence the allergenicity of Aspergillus fumigatus

Increased susceptibility to allergies has been documented in the Western world in recent decades. However, a comprehensive understanding of its causes is not yet available. It is therefore essential to understand trends and mechanisms of allergy‐inducing agents, such as fungal conidia. In this study, we investigated the hypothesis that environmental conditions linked to global atmospheric changes can affect the allergenicity of Aspergillus fumigatus, a common allergenic fungal species in indoor and outdoor environments and in airborne particulate matter. We show that fungi grown under present‐day CO₂ levels (392 ppm) exhibit 8.5 and 3.5 fold higher allergenicity compared to fungi grown at preindustrial (280 ppm) and double (560 ppm) CO₂ levels, respectively. A corresponding trend is observed in the expression of genes encoding for known allergenic proteins and in the major allergen Asp f1 concentrations, possibly due to physiological changes such as respiration rates and the nitrogen content of the fungus, influenced by the CO₂ concentrations. Increased carbon and nitrogen levels in the growth medium also lead to a significant increase in the allergenicity. We propose that climatic changes such as increasing atmospheric CO₂ levels and changes in the fungal growth medium may impact the ability of allergenic fungi such as A. fumigatus to induce allergies.