A Burkholderia cenocepacia MurJ (MviN) homolog is essential for cell wall peptidoglycan synthesis and bacterial viability

The cell wall peptidoglycan (PG) of Burkholderia cenocepacia, an opportunistic pathogen, has not yet been characterized. However, the B. cenocepacia genome contains homologs of genes encoding PG biosynthetic functions in other bacteria. PG biosynthesis involves the formation of the undecaprenyl-pyrophosphate-linked N-acetyl glucosamine-N-acetyl muramic acid-pentapeptide, known as lipid II, which is built on the cytosolic face of the cell membrane. Lipid II is then translocated across the membrane and its glycopeptide moiety becomes incorporated into the growing cell wall mesh; this translocation step is critical to PG synthesis. We have investigated candidate flippase homologs of the MurJ family in B. cenocepacia. Our results show that BCAL2764, herein referred to as murJBc, is indispensable for viability. Viable B. cenocepacia could only be obtained through a conditional mutagenesis strategy by placing murJBc under the control of a rhamnose-inducible promoter. Under rhamnose depletion, the conditional strain stopped growing and individual cells displayed morphological abnormalities consistent with a defect in PG synthesis. Bacterial cells unable to express MurJBc underwent cell lysis, while partial MurJBc depletion sensitized the mutant to the action of β-lactam antibiotics. Depletion of MurJBc caused accumulation of PG precursors consistent with the notion that this protein plays a role in lipid II flipping to the periplasmic compartment. Reciprocal complementation experiments of conditional murJ mutants in B. cenocepacia and Escherichia coli with plasmids expressing MurJ from each strain indicated that MurJBc and MurJEc are functional homologs. Together, our results are consistent with the notion that MurJBc is a PG lipid II flippase in B. cenocepacia.

Human mesenchymal stem cells reduce the severity of acute lung injury in a sheep model of bacterial pneumonia

Background Human bone marrow-derived mesenchymal stem (stromal) cells (hMSCs) improve survival in mouse models of acute respiratory distress syndrome (ARDS) and reduce pulmonary oedema in a perfused human lung preparation injured with Escherichia coli bacteria. We hypothesised that clinical grade hMSCs would reduce the severity of acute lung injury (ALI) and would be safe in a sheep model of ARDS.

Methods Adult sheep (30–40 kg) were surgically prepared. After 5 days of recovery, ALI was induced with cotton smoke insufflation, followed by instillation of live Pseudomonas aeruginosa (2.5×10¹¹ CFU) into both lungs under isoflurane anaesthesia. Following the injury, sheep were ventilated, resuscitated with lactated Ringer's solution and studied for 24 h. The sheep were randomly allocated to receive one of the following treatments intravenously over 1 h in one of the following groups: (1) control, PlasmaLyte A, n=8; (2) lower dose hMSCs, 5×10⁶ hMSCs/kg, n=7; and (3) higher-dose hMSCs, 10×10⁶ hMSCs/kg, n=4.

Results By 24 h, the PaO₂/FiO₂ ratio was significantly improved in both hMSC treatment groups compared with the control group (control group: PaO₂/FiO₂ of 97±15 mm Hg; lower dose: 288±55 mm Hg (p=0.003); higher dose: 327±2 mm Hg (p=0.003)). The median lung water content was lower in the higher-dose hMSC-treated group compared with the control group (higher dose: 5.0 g wet/g dry [IQR 4.9–5.8] vs control: 6.7 g wet/g dry [IQR 6.4–7.5] (p=0.01)). The hMSCs had no adverse effects.

Conclusions Human MSCs were well tolerated and improved oxygenation and decreased pulmonary oedema in a sheep model of severe ARDS.

Towards an understanding of bacterial metabolites prodigiosin and violacein and their potential for use in commercial sunscreens

Synopsis
Objectives

To exploit the microbial ecology of bacterial metabolite production and, specifically, to: (i) evaluate the potential use of the pigments prodigiosin and violacein as additives to commercial sunscreens for protection of human skin, and (ii) determine antioxidant and antimicrobial activities (against pathogenic bacteria) for these two pigments.

Methods
Prodigiosin and violacein were used to supplement extracts of Aloe vera leaf and Cucumis sativus (cucumber) fruit which are known to have photoprotective activity, as well as some commercial sunscreen preparations. For each, sunscreen protection factors (SPFs) were determined spectrophotometrically. Assays for antimicrobial activity were carried out using 96-well plates to quantify growth inhibition of Staphylococcus aureus and Escherichia coli.
Results
For the plant extracts, SPFs were increased by an order of magnitude (i.e. up to ~3.5) and those for the commercial sunscreens increased by 10–22% (for 4% w/w violacein) and 20–65% (for 4% w/w prodigiosin). The antioxidant activities of prodigiosin and violacein were approximately 30% and 20% those of ascorbic acid (a well-characterized, potent antioxidant). Violacein inhibited S. aureus (IC_{506.99 ± 0.146 μM) but not E. coli, whereas prodigiosin was effective against both of these bacteria (IC50 values were 0.68 ± 0.06 μM and 0.53 ± 0.03 μM, respectively).}

Conclusion
The bacterial pigments prodigiosin and violacein exhibited antioxidant and antimicrobial activities and were able to increase the SPF of commercial sunscreens as well as the extracts of the two plant species tested. These pigments have potential as ingredients for a new product range of and, indeed, represent a new paradigm for sunscreens that utilize substances of biological origin. We discussed the biotechnological potential of these bacterial metabolites for use in commercial sunscreens, and the need for studies of mammalian cells to determine safety.

Modeling study of woody biomass:Interactions of cellulose, hemicellulose, and lignin

Lignocellulosic biomass pretreatment and the subsequent thermal conversion processes to produce solid, liquid, and gas biofuels are attractive solutions for today's energy challenges. The structural study of the main components in biomass and their macromolecular complexes is an active and ongoing research topic worldwide. The interactions among the three main components, cellulose, hemicellulose, and lignin, are studied in this paper using electronic structure methods, and the study includes examining the hydrogen bond network of cellulose-hemicellulose systems and the covalent bond linkages of hemicellulose-lignin systems. Several methods (semiempirical, Hartree-Fock, and density functional theory) using different basis sets were evaluated. It was shown that theoretical calculations can be used to simulate small model structures representing wood components. By comparing calculation results with experimental data, it was concluded that B3LYP/6-31G is the most suitable basis set to describe the hydrogen bond system and B3LYP/6-31G(d,p) is the most suitable basis set to describe the covalent system of woody biomass. The choice of unit model has a much larger effect on hydrogen bonding within cellulose-hemicellulose system, whereas the model choice has a minimal effect on the covalent linkage in the hemicellulose-lignin system. © 2011 American Chemical Society.

Formation mechanism of levoglucosan and formaldehyde during cellulose pyrolysis

Biomass pyrolysis is an efficient way to transform raw biomass or organic waste materials into useable energy, including liquid, solid, and gaseous materials. Levoglucosan (1,6-anhydro-β-d-glucopyranose) and formaldehyde are two important products in biomass pyrolysis. The formation mechanism of these two products was investigated using the density functional theory (DFT) method based on quantum mechanics. It was found that active anhydroglucose can be obtained from a cellulose homolytic reaction during high-temperature steam gasification of the biomass process. Anhydroglucose undergoes a hydrogen-donor reaction and forms an intermediate, which can transform into the products via three pathways, one (path 1) for the formation of levoglucosan and two (paths 2 and 3) for formaldehyde. A total of six elementary reactions are involved. At a pressure of 1 atm, levoglucosan can be formed at all of the temperatures (450-750 K) considered in this simulation, whereas formaldehyde can be formed only when the temperature is higher than 475 K. Moreover, the energy barrier of levoglucosan formation is lower than that of formaldehyde, which is in agreement with the mechanism proposed in the experiments. © 2011 American Chemical Society.

Kinetics of levoglucosan and formaldehyde formation during cellulose pyrolysis process

The mechanisms and kinetics studies of the formation of levoglucosan and formaldehyde from anhydroglucose radical have been carried out theoretically in this paper. The geometries and frequencies of all the stationary points are calculated at the B3LYP/6-31+G(D,P) level based on quantum mechanics, Six elementary reactions are found, and three global reactions are involved. The variational transition-state rate constants for the elementary reactions are calculated within 450-1500 K. The global rate constants for every pathway are evaluated from the sum of the individual elementary reaction rate constants. The first-order Arrhenius expressions for these six elementary reactions and the three pathways are suggested. By comparing with the experimental data, computational methods without tunneling correction give good description for Path1 (the formation of levoglucosan); while methods with tunneling correction (zero-curvature tunneling and small-curvature tunneling correction) give good results for Path2 (the first possibility for the formation of formaldehyde), all the test methods give similar results for Path3 (the second possibility for the formation of formaldehyde), all the modeling results for Path3 are in good agreement with the experimental data, verifying that it is the most possible way for the formation of formaldehyde during cellulose pyrolysis. © 2012 Elsevier Ltd. All rights reserved.

Thermal decomposition mechanism of levoglucosan during cellulose pyrolysis

Levoglucosan (1,6-anhydro-β-d-glucopyranose) decomposition is an important step during cellulose pyrolysis and for secondary tar reactions. The mechanism of levoglucosan thermal decomposition was studied in this paper using density functional theory methods. The decomposition included direct CO bond breaking, direct CC bond breaking, and dehydration. In total, 9 different pathways, including 16 elementary reactions, were studied, in which levoglucosan serves as a reactant. The properties of the reactants, transition states, intermediates, and products for every elementary reaction were obtained. It was found that 1-pentene-3,4-dione, acetaldehyde, 2,3-dihydroxypropanal, and propanedialdehyde can be formed from the CO bond breaking decomposition reactions. 1,2-Dihydroxyethene and hydroxyacetic acid vinyl ester can be formed from the CC bond breaking decomposition reactions. It was concluded that CO bond breaking is easier than CC bond breaking due to a lower activation energy and a higher released energy. During the 6 levoglucosan dehydration pathways, one water molecule which composed of a hydrogen atom from C3 and a hydroxyl group from C2 is the preferred pathway due to a lower activation energy and higher product stability. © 2012 Elsevier B.V. All rights reserved.

Kinetics study on thermal dissociation of levoglucosan during cellulose pyrolysis

(Chemical Equation Presented) The mechanisms and kinetics studies of the levoglucosan (LG) primary decomposition during cellulose pyrolysis have been carried out theoretically in this paper. Three decomposition mechanisms (C-O bond scission, C-C bond scission, and LG dehydration) including nine pathways and 16 elementary reactions were studied at the B3LYP/6-31 + G(D,P) level based on quantum mechanics. The variational transi-tion- state rate constants for every elementary reaction and every pathway were calculated within 298-1550 K. The first-order Arrhenius expressions for these 16 elementary reactions and nine pathways were suggested. It was concluded that computational method using transition state theory (TST) without tunneling correction gives good description for LG decomposition by comparing with the experimental result. With the temperature range of 667-1327 K, one dehydration pathway, with one water molecule composed of a hydrogen atom from C3 and a hydroxyl group from C2, is a preferred LG decomposition pathway by fitting well with the experimental results. The calculated Arrhenius plot of C-O bond scission mechanism is better agreed with the experimental Arrhenius plot than that of C-C bond scission. This C-O bond scission mechanism starts with breaking of C1-O5 and C6-O1 bonds with formation of CO molecule (C1-O1) simultaneously. C-C bond scission mechanism is the highest energetic barrier pathway for LG decomposition. © 2013 Elsevier Ltd. All rights reserved.

Levoglucosan formation mechanisms during cellulose pyrolysis

Levoglucosan is one important primary product during cellulose pyrolysis either as an intermediate or as a product. Three available mechanisms for levoglucosan formation have been studied theoretically in this paper, which are free-radical mechanism; glucose intermediate mechanism; and levoglucosan chain-end mechanism. All the elementary reactions included in the pathway of every mechanism were investigated; thermal properties including activation energy, Gibbs free energy, and enthalpy for every pathway were also calculated. It was concluded that free-radical mechanism has the highest energy barrier during the three levoglucosan formation mechanisms, glucose intermediate mechanism has lower energy barrier than free-radical mechanism, and levoglucosan chain-end mechanism is the most reasonable pathway because of the lowest energy barrier. By comparing with the activation energy obtained from the experimental results, it was also concluded that levoglucosan chain-end mechanism fits better with the experimental data for the formation of levoglucosan. © 2013 Elsevier B.V. All rights reserved.

S21 Culture Independent Identification Of Bacterial Communities In The Respiratory Tract Of Patients With Copd, Healthy Non-smokers And Healthy Smokers

Introduction and aims: The role bacteria play in the development and progression of Chronic Obstructive Pulmonary Disease (COPD) is unclear. We used culture-independent methods to describe differences and/or similarities in microbial communities in the lower airways of patients with COPD, healthy non-smokers and smokers.

Methods: Bronchial wash samples were collected from patients with COPD (GOLD 1–3; n = 18), healthy non-smokers (HV; n = 11) and healthy smokers (HS; n = 8). Samples were processed using the Illumina MiSeq platform. The Shannon-Wiener Index (SW) of diversity, lung obstruction (FEV1/FVC ratio) and ordination by Non-Metric Multidimensional Scaling (NMDS) on Bray-Curtis dissimilarity indices were analysed to evaluate how samples were related. Principal component analysis (PCA) was performed to assess the effect specific taxa had within each cohort. Characteristics of each cohort are shown in Table 1.

Results: There was no difference in taxa richness between cohorts (range: 69–71; p = 0.954). Diversity (SW Index) was significantly lower in COPD samples compared to samples from HV and HS (p = 0.009 and p = 0.033, respectively). There was no significant difference between HV and HS (p = 0.186). The FEV1/FVC ratio was significantly lower for COPD compared to HV (p = 9*10–8) and HS (p = 2*10–6), respectively. NMDS analysis showed that communities belonging to either of the healthy groups were more similar to each other than they were to samples belonging to the COPD group. PCA analysis showed that members of Streptococcus sp. and Haemophilus sp. had the largest effect on the variance explained in COPD. In HS, Haemophilus sp., Fusobaterium sp., Actinomyces sp., Prevotella sp. and Veillonella sp. had the largest effect on the variance explained, while in HV Neisseria sp., Porphyromonas sp., Actinomyces sp., Atopobium sp., Prevotella and Veillonella sp. had the largest effect on the variance explained.

Conclusions: The study demonstrates that microbial communities in the lower airways of patients with COPD are significantly different from that seen in healthy comparison groups. Patients with COPD had lower microbial diversity than either of the healthy comparison groups, higher relative abundance of members of Streptococcus sp. and lower relative abundance of a number of key anaerobes.Characteristics