A novel method of quantifying root exudation in the presence of soil microflora

A microcosm is described in which root exudation may be estimated in the presence of microorganisms. Ryegrass seedlings are grown in microcosms in which roots were spatially separated from a microbial inoculant by a Millipore membrane. Seedlings grown in the microcosms were labelled with [¹⁴C]-CO₂, and the fate of the label within the plant and rhizosphere was determined. Inoculation of the microcosms with Cladosporium resinae increased net fixation of the [¹⁴C] label compared to plants grown under sterile conditions. Inoculation also increased root exudation. The use of the microcosm was illustrated and its applications discussed. © 1991 Kluwer Academic Publishers.

Assessment of Anaerobic Biodegradation of Aromatic Hydrocarbons: The Impact of Molecular Biology Approaches

One of the most cost effective methods of pollution remediation is through natural attenuation where the resident microorganisms are responsible for the breakdown of pollutants (Dou et al. 2008). Other forms of bioremediation - such as analogue enrichment, composting and bio-venting - also use the microbes already present in a contaminated site to enhance the remediation process. In order for these approaches to be successful, in an industrial setting, some form of monitoring needs to take place enabling conclusions to be drawn about the degradation processes occurring. In this review we look at some key molecular biology techniques that have the potential to act as a monitoring tool for industries dealing with contaminated land. 

Secure Switch-and-Stay Combining (SSSC) for Cognitive Relay Networks

In this paper, we study a two-phase underlay cognitive relay network, where there exists an eavesdropper who can overhear the message. The secure data transmission from the secondary source to secondary destination is assisted by two decode-and-forward (DF) relays. Although the traditional opportunistic relaying technique can choose one relay to provide the best secure performance, it needs to continuously have the channel state information (CSI) of both relays, and may result in a high relay switching rate. To overcome these limitations, a secure switch-and-stay combining (SSSC) protocol is proposed where only one out of the two relays is activated to assist the secure data transmission, and the secure relay switching occurs when the relay cannot support the secure communication any longer. This security switching is assisted by either instantaneous or statistical eavesdropping CSI. For these two cases, we study the system secure performance of SSSC protocol, by deriving the analytical secrecy outage probability as well as an asymptotic expression for the high main-to-eavesdropper ratio (MER) region. We show that SSSC can substantially reduce the system complexity while achieving or approaching the full diversity order of opportunistic relaying in the presence of the instantaneous or statistical eavesdropping CSI.

Tyrosine phosphorylation and dephosphorylation in Burkholderia cenocepacia affect biofilm formation, growth under nutritional deprivation, and pathogenicity

Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important post-translational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF, and the low molecular weight protein tyrosine phosphatases BCAM0208, BceD and BCAL2200 of B. cenocepacia K56-2. We show that BCAM1331, BceF, BCAM0208 and BceD contributed to biofilm formation, while BCAL2200 was required for growth in nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low molecular weight protein tyrosine phosphatases genes resulted in attenuation of B. cenocepacia intramacrophage survival and reduced pathogenicity in the Galleria mellonella larvae infection model. Experimental evidence indicates that BCAM1331 displays a reduced
tyrosine autophosphorylation activity compared to BceF. Using the artificial substrate p-nitrophenyl phosphate, the phosphatase activity of the three low molecular weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 becomes tyrosine phosphorylated in vivo and catalyzes its auto-dephosphorylation. Together, our data suggest that despite having similar biochemical activities low molecular weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of B. cenocepacia.

Effects of LL-37 on Human Gingival Fibroblast Function

Background and Objectives: Gingival fibroblasts play a significant role in the innate immune response of the periodontium to bacterial stimulation. A number of microorganisms and their by-products induce a host response that commonly leads to tissue destruction and periodontal disease progression. LL-37 is an antimicrobial peptide which has multiple roles in host defence including immunomodulation and wound-healing. We have investigated the role of LL-37 on the responsiveness of human gingival fibroblasts to microbial challenge from E. coli lipopolysaccharide (LPS) and P. gingivalis LPS, as well as exploring the direct effects of LL-37 on human gingival fibroblasts. Methods: The effect of LL-37 on bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. The influence of LL-37 on bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot. The direct effects of LL-37 on modulating gingival fibroblasts gene expression were initially determined by DNA microarray analysis and subsequently confirmed by quantitative polymerase chain reaction (Q-PCR) and ELISA analysis of 9 selected genes. Results: Bacterial LPS-induced IL-8 and IL-6 production by human gingival fibroblasts were significantly reduced in the presence of LL-37 at concentrations in the range of 1-10 µg/ml (p<0.05). The presence of LL-37 at a concentration of 5 µg/ml led to a reduction in LPS-induced IκBα degradation by E. coli LPS (100 ng/ml) and P. gingivalis LPS (10 µg/ml). LL-37 (50 µg/ml) significantly altered the gene expression of 367 genes in human gingival fibroblasts by at least 2-fold. CXCL1, CXCL2, CXCL3, IL-24, IL-8, CCL2, and SOCS3 mRNA were significantly upregulated by LL-37 (p<0.05). LL-37 also significantly stimulated expression of IL-8, hepatocyte growth factor (HGF) and CXCL1 (p<0.05) at the protein level. Discussion: LL-37 plays an important role in the innate immune response due to its broad spectrum antimicrobial and immunomodulatory activity. The ability of LL-37 to directly regulate expression of a range of genes, central to the pathogenesis of periodontitis, identifies multiple roles for the peptide in host homeostasis.

Protective role of glycerol against benzene stress: insights from the Pseudomonas putida proteome

Chemical activities of hydrophobic substances can determine the windows of environmental conditions over which microbial systems function and the metabolic inhibition of microorganisms by benzene and other hydrophobes can, paradoxically, be reduced by compounds that protect against cellular water stress (Bhaganna et al. in Microb Biotechnol 3:701-716, 2010; Cray et al. in Curr Opin Biotechnol 33:228-259, 2015a). We hypothesized that this protective effect operates at the macromolecule structure-function level and is facilitated, in part at least, by genome-mediated adaptations. Based on proteome profiling of the soil bacterium Pseudomonas putida, we present evidence that (1) benzene induces a chaotrope-stress response, whereas (2) cells cultured in media supplemented with benzene plus glycerol were protected against chaotrope stress. Chaotrope-stress response proteins, such as those involved in lipid and compatible-solute metabolism and removal of reactive oxygen species, were increased by up to 15-fold in benzene-stressed cells relative to those of control cultures (no benzene added). By contrast, cells grown in the presence of benzene + glycerol, even though the latter grew more slowly, exhibited only a weak chaotrope-stress response. These findings provide evidence to support the hypothesis that hydrophobic substances induce a chaotropicity-mediated water stress, that cells respond via genome-mediated adaptations, and that glycerol protects the cell's macromolecular systems. We discuss the possibility of using compatible solutes to mitigate hydrocarbon-induced stresses in lignocellulosic biofuel fermentations and for industrial and environmental applications.

Genome Expression Profiling-Based Identification and Administration Efficacy of Host-Directed Antimicrobial Drugs against Respiratory Infection by Nontypeable Haemophilus influenzae

Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.

Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus

The interaction between microorganisms and host defense mechanisms is a decisive factor for the survival of marine bivalves. They rely on cell-mediated and humoral reactions to overcome the pathogens that naturally occur in the marine environment. In order to understand host defense reactions in animals inhabiting extreme environments we investigated some of the components from the immune system of the deep sea hydrothermal vent mussel Bathymodiolus azoricus. Cellular constituents in the hemolymph and extrapallial fluid were examined and led to the identification of three types of hemocytes revealing the granulocytes as the most abundant type of cell. To further characterize hemocyte types, the presence of cell surface carbohydrate epitopes was demonstrated with fluorescent WGA lectin, which was mostly ascribed to the granulocytes. Cellular reactions were then investigated by means of phagocytosis and by the activation of putative MAPKs using the microbial compounds zymosan, glucan, peptidoglycan and lipopolysaccharide. Two bacterial agents, Bacillus subtilis and Vibrio parahaemolyticus, were also used to stimulate hemocytes. The results showed that granulocytes were the main phagocytic cells in both hemolymph and extrapallial fluid of B. azoricus. Western blotting analyses using commercially available antibodies against ERK, p38 and JNK, suggested that these putative kinases are involved in signal transduction pathways during experimental stimulation of B. azoricus hemocytes. The fluorescent Ca²⁺ indicator Fura-2 AM was also insightful in demonstrating hemocyte stimulation in the presence of laminarin or live V. parahaemolyticus. Finally, the expression of the antibacterial gene mytilin was analyzed in gill tissues by means of RT-PCR and whole-mount in situ hybridization. Mytilin transcripts were localized in hemocytes underlying gill epithelium. Moreover, mytilin was induced by exposure of live animals to V. parahaemolyticus. These findings support the premise of a conserved innate immune system in B. azoricus. Such system is comparable to other Bivalves and involves the participation of cellular and humoral components. © 2008 Elsevier Inc. All rights reserved.

Mineralogy, chemistry and biological contingents of an early-middle Miocene Antarctic paleosol and its relevance as a Martian analogue

Fossil mesofauna and bacteria recovered from a paleosol in a moraine situated adjacent to the inland ice, Antarctica, and dating to the earliest glacial event in the Antarctic Dry Valleys opens several questions. The most important relates to understanding of the mineralogy and chemistry of the weathered substrate habitat in which Coleoptera apparently thrived at some point in the Early/Middle Miocene and perhaps earlier. Here, Coleoptera remains are only located in one of six horizons in a paleosol formed in moraine deposited during the alpine glacial event (> 15 Ma). A tendency for quartz to decrease upward in the section may be a detrital effect or a product of dissolution in the early stage of profile morphogenesis when climate was presumably milder and the depositing glacier of temperate type. Discontinuous distributions of smectite, laumontite, and hexahydrite may have provided nutrients and water to mesofauna and bacteria during the early stage of biotic colonization of the profile. Because the mesofauna were members of burrowing Coleoptera species, future work should assess the degree to which the organisms occupied other sites in the Dry Valleys in the past. Whereas there is no reasonable expectations of finding Coleoptera/insect remains on Mars, the chemistry and mineralogy of the paleosol is within a life expectancy window for the presence of microorganisms, principally bacteria and fungi. Thus, parameters discussed here within this Antarctic paleosol could provide an analogue to identifying similar fossil or life-bearing weathered regolith on Mars.

Industrial scale microwave processing of tomato juice using a novel continuous microwave system

This study evaluated the effect of an industrial scale continuous flow microwave volumetric heating system in comparison to conventional commercial scale pasteurisation for the processing of tomato juice in terms of physicochemical properties, microbial characteristics and antioxidant capacity. The effect against oxidative stress in Caco-2 cells, after in vitro digestion was also investigated. Physicochemical and colour characteristics of juices were very similar between technologies and during storage. Both conventional and microwave pasteurisation inactivated microorganisms and kept them in low levels throughout storage. ABTS⁺ values, but not ORAC, were higher for the microwave pasteurised juice at day 0 however no significant differences between juices were observed during storage. Juice processed with the microwave system showed an increased cytoprotective effect against H₂O₂ induced oxidation in Caco-2 cells. Organoleptic analysis revealed that the two tomato juices were very similar. The continuous microwave volumetric heating system appears to be a viable alternative to conventional pasteurisation.

Antifungal Activity of LL-37 and Truncated Mimetics

Background: Candida albicans is a commensal organism and a constituent of the normal oral flora. Cell concentrations of 1x102 cells/ml and below are indicative of commensal colonisation in the oral cavity, above this level C. albicans can become an opportunistic pathogen; it is the most prevalent human fungal pathogen and a causal agent of the oral infection, candidiasis. The capacity of C. albicans to cause infection arises from its ability to exist in a biofilm ecosystem. Mature C. albicans biofilms display a high level of resistance to antifungals and the need for other therapeutic options has become paramount. Objectives: The objectives of the current study were to determine the antifungal activity of LL-37 (a member of the human cathelicidin family) and two truncated peptide mimetics against C. albicans in both planktonic and biofilm form. Methods: Radial diffusion assays were used to obtain the minimum inhibitory concentration (MIC) of LL-37 and the truncated mimetics KE-18 and KR-12 against planktonic C. albicans. A 96 well microtitre plate assay was employed to study the effects of the peptides on early candida biofilm formation (up to 24 hours) compared with the antifungal drug fluconazole. Biofilm quantification was achieved using the crystal violet assay. Results: MIC values obtained: LL-37 >250µg/ml; KE-18 51µg/ml; and KR-12 11µg/ml. LL-37 significantly reduced the quantity of biofilm formed by C.albicans at both the 4 h and 24 h timepoints (p <0.0001). KE-18 showed significant biofilm reduction over 4 h and 24 h (p=0.0002, p=0.013 respectively), KR-12 showed significant reduction at the 24 h time point only (p=0.0256). Conclusions: Results suggest that LL-37 has the ability to disrupt early biofilm formation of C. albicans with its potency of action similar with that of fluconazole.

Pathogens in Milk: Enterobacter Species. Reference Module in Food Sciences.

Enterobacter species commonly occur in the environment and are recognized as opportunistic human pathogens in clinical settings. However, with the exception of Enterobacter sakazakii (Cronobacter), Enterobacter species are not normally considered foodborne pathogens. Cronobacter are particularly associated with illness in infants, particularly within the first 3 months after birth. Therefore, although Cronobacter are found in a wide range of fresh and dried food materials, it is their contamination of the infant formula production chain that is the major cause for concern. Cronobacter are noted for their ability to survive during desiccation and their persistence in dried infant food for at least 2 years.

A Burkholderia Type VI Effector Deamidates Rho GTPases to Activate the Pyrin Inflammasome

Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome.