Bacterial membrane injuries induced by lactacin F and nisin

The combined action of nisin and lactacin F, two bacteriocins produced by lactic acid bacteria, is additive. In this report, the basis of this effect is examined. Channels formed by lactacin F were studied by experiments using planar lipid bilayers, and bactericidal effects were analyzed by flow cytometry. Lactacin F produced pores with a conductance of 1 ns in black lipid bilayers in 1 mM KClat 10 mV at 20°C. Pore formation was strongly dependent on voltage. Although lactacin F formed pores at very low potential (10 mV), the dependence was exponentialabov e 40 mV. The injuries induced by nisin and lactacin F in the membranes of Lactobacillus helveticus produced different flow cytometric profiles. Probably, when both bacteriocins are present, each acts separately; their cooperation may be due to an increase in the number of single membrane injuries

Membrane interaction of polymyxin B and synthetic analogues studied in biomimetic systems: implications for antibacterial action

Membrane-active antimicrobial peptides, such as polymyxin B (PxB), are currently in the spotlight as potential candidates toovercome bacterial resistance. We have designed synthetic analogs ofPxB in order to determine the structural requirements for membraneaction. Since the mechanism of action of PxB involves interaction withboth the outer membrane and the cytoplasmic membrane of Gramnegative bacteria, we have used an approach based on mimicking theouter layers of these membranes using monolayers, Langmuir-Blodgettfilms and unilamelar vesicles, and applying a battery of biophysicalmethods in order to dissect the different events of membraneinteraction. Collectively, results indicate that the PxB analogues act inthe bacterial membrane by the same mechanism than PxB, and that cationic amphipathicity determines peptide activity.

Membrane interaction of polymyxin B and synthetic analogues studied in biomimetic systems: implications for antibacterial action

Membrane-active antimicrobial peptides, such as polymyxin B (PxB), are currently in the spotlight as potential candidates toovercome bacterial resistance. We have designed synthetic analogs ofPxB in order to determine the structural requirements for membraneaction. Since the mechanism of action of PxB involves interaction withboth the outer membrane and the cytoplasmic membrane of Gramnegative bacteria, we have used an approach based on mimicking theouter layers of these membranes using monolayers, Langmuir-Blodgettfilms and unilamelar vesicles, and applying a battery of biophysicalmethods in order to dissect the different events of membraneinteraction. Collectively, results indicate that the PxB analogues act inthe bacterial membrane by the same mechanism than PxB, and that cationic amphipathicity determines peptide activity.

Membrane interaction of a new synthetic antimicrobial lipopetide sp-85 with broad spectrum activity

Antimicrobial peptides offer a new class of therapeutic agents to which bacteria may not be able todevelop genetic resistance, since their main activity is in the lipid component of the bacterial cell mem-brane. We have developed a series of synthetic cationic cyclic lipopeptides based on natural polymyxin,and in this work we explore the interaction of sp-85, an analog that contains a C12 fatty acid at theN-terminus and two residues of arginine. This analog has been selected from its broad spectrum antibac-terial activity in the micromolar range, and it has a disruptive action on the cytoplasmic membrane ofbacteria, as demonstrated by TEM. In order to obtain information on the interaction of this analog withmembrane lipids, we have obtained thermodynamic parameters from mixed monolayers prepared withPOPG and POPE/POPG (molar ratio 6:4), as models of Gram positive and Gram negative bacteria, respec-tively. LangmuirBlodgett films have been extracted on glass plates and observed by confocal microscopy,and images are consistent with a strong destabilizing effect on the membrane organization induced bysp-85. The effect of sp-85 on the membrane is confirmed with unilamelar lipid vesicles of the same com-position, where biophysical experiments based on fluorescence are indicative of membrane fusion andpermeabilization starting at very low concentrations of peptide and only if anionic lipids are present.Overall, results described here provide strong evidence that the mode of action of sp-85 is the alterationof the bacterial membrane permeability barrier.

Dual Action of BPC194: A Membrane Active Peptide Killing Bacterial Cells

Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Förster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis

Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics

Membrane-aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long-term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real-time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10-fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r-strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal

Outer membrane vesicles from cold-adapted antarctic bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter with potential biotechnological applications. Transmission electron microscopy (TEM) analysis after high-pressure freezing and freeze substitution (HPF-FS) showed that this extracellular matter is structurally complex, appearing around cells as a netlike mesh, and composed of an exopolymeric substance (EPS) containing large numbers of outer membrane vesicles (OMVs). Isolation, purification and protein profiling via 1D SDS-PAGE confirmed the outer membrane origin of these Antarctic bacteria OMVs. In an initial attempt to elucidate the role of OMVs in cold-adapted strains of Gram-negative bacteria, a proteomic analysis demonstrated that they were highly enriched in outer membrane proteins and periplasmic proteins associated with nutrient processing and transport, suggesting that the OMVs may be involved in nutrient sensing and bacterial survival. OMVs from Gram-negative bacteria are known to play a role in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. A structural study of Shewanella vesiculosa M7T using TEM and Cryo-TEM revealed that this Antarctic Gram-negative bacterium naturally releases conventional one-bilayer OMVs, together with a more complex type of OMV, previously undescribed, which on formation drags along inner membrane and cytoplasmic content and can therefore also entrap DNA.

Outer membrane vesicles from cold-adapted antarctic bacteria

Many Gram-negative, cold-adapted bacteria from the Antarctic environment produce large amounts of extracellular matter with potential biotechnological applications. Transmission electron microscopy (TEM) analysis after high-pressure freezing and freeze substitution (HPF-FS) showed that this extracellular matter is structurally complex, appearing around cells as a netlike mesh, and composed of an exopolymeric substance (EPS) containing large numbers of outer membrane vesicles (OMVs). Isolation, purification and protein profiling via 1D SDS-PAGE confirmed the outer membrane origin of these Antarctic bacteria OMVs. In an initial attempt to elucidate the role of OMVs in cold-adapted strains of Gram-negative bacteria, a proteomic analysis demonstrated that they were highly enriched in outer membrane proteins and periplasmic proteins associated with nutrient processing and transport, suggesting that the OMVs may be involved in nutrient sensing and bacterial survival. OMVs from Gram-negative bacteria are known to play a role in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. A structural study of Shewanella vesiculosa M7T using TEM and Cryo-TEM revealed that this Antarctic Gram-negative bacterium naturally releases conventional one-bilayer OMVs, together with a more complex type of OMV, previously undescribed, which on formation drags along inner membrane and cytoplasmic content and can therefore also entrap DNA.

Individual-based modelling of bacterial cultures to study the lag ohase

Els bacteris són la forma dominant de vida del planeta: poden sobreviure en medis molt adversos, i en alguns casos poden generar substàncies que quan les ingerim ens són tòxiques. La seva presència en els aliments fa que la microbiologia predictiva sigui un camp imprescindible en la microbiologia dels aliments per garantir la seguretat alimentària. Un cultiu bacterià pot passar per quatre fases de creixement: latència, exponencial, estacionària i de mort. En aquest treball s’ha avançat en la comprensió dels fenòmens intrínsecs a la fase de latència, que és de gran interès en l’àmbit de la microbiologia predictiva. Aquest estudi, realitzat al llarg de quatre anys, s’ha abordat des de la metodologia Individual-based Modelling (IbM) amb el simulador INDISIM (INDividual DIScrete SIMulation), que ha estat millorat per poder fer-ho. INDISIM ha permès estudiar dues causes de la fase de latència de forma separada, i abordar l’estudi del comportament del cultiu des d’una perspectiva mesoscòpica. S’ha vist que la fase de latència ha de ser estudiada com un procés dinàmic, i no definida per un paràmetre. L’estudi de l’evolució de variables com la distribució de propietats individuals entre la població (per exemple, la distribució de masses) o la velocitat de creixement, han permès distingir dues etapes en la fase de latència, inicial i de transició, i aprofundir en la comprensió del que passa a nivell cel•lular. S’han observat experimentalment amb citometria de flux diversos resultats previstos per les simulacions. La coincidència entre simulacions i experiments no és trivial ni casual: el sistema estudiat és un sistema complex, i per tant la coincidència del comportament al llarg del temps de diversos paràmetres interrelacionats és un aval a la metodologia emprada en les simulacions. Es pot afirmar, doncs, que s’ha verificat experimentalment la bondat de la metodologia INDISIM.

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.

Mathematical description of bacterial traveling pulses

The Keller-Segel system has been widely proposed as a model for bacterial waves driven by chemotactic processes. Current experiments on E. coli have shown precise structure of traveling pulses. We present here an alternative mathematical description of traveling pulses at a macroscopic scale. This modeling task is complemented with numerical simulations in accordance with the experimental observations. Our model is derived from an accurate kinetic description of the mesoscopic run-and-tumble process performed by bacteria. This model can account for recent experimental observations with E. coli. Qualitative agreements include the asymmetry of the pulse and transition in the collective behaviour (clustered motion versus dispersion). In addition we can capture quantitatively the main characteristics of the pulse such as the speed and the relative size of tails. This work opens several experimental and theoretical perspectives. Coefficients at the macroscopic level are derived from considerations at the cellular scale. For instance the stiffness of the signal integration process turns out to have a strong effect on collective motion. Furthermore the bottom-up scaling allows to perform preliminary mathematical analysis and write efficient numerical schemes. This model is intended as a predictive tool for the investigation of bacterial collective motion.

Efecto de los contenidos de C, Mn y Si en el comportamiento a fluencia en caliente de aceros de construcción al carbono y microaleados: aplicación a la obtención de grano ultrafino en productos largos laminados

Després d’aplicar alguns tractaments d’elaboració i conservació als aliments, queden bacteris lesionats. Aquests bacteris perden la capacitat de créixer en els medis de cultiu selectiu convencionals, de manera que se’n subestima el recompte. Malgrat això, poden recuperar-se als aliments i suposar un risc per la salut, ja que alguns encara poden mantenir activitat metabòlica i integritat estructural. En aquest projecte, es van optimitzar protocols de preparació de mostres per citometria de flux (CF) per avaluar l’estat fisiològic de patògens alimentaris (Escherichia coli O157:H7, Salmonella Enteritidis i Listeria monocytogenes) sotmesos a estrès. Es van estudiar principalment dos paràmetres fisiològics: la integritat de membrana, mitjançant iodur de propidi i fluorocroms de la família SYTO; i l’activitat respiratòria, per la reducció intracel•lular d’una sal de tetrazole, el CTC. En primer lloc, es van avaluar variables de protocol, com la concentració de colorant, la ràtio entre colorants, la solució de tinció i el temps d’incubació, en mostres control (cèl•lules sanes i mortes). A continuació, els protocols optimitzats es van aplicar a suspensions bacterianes en medi de cultiu que prèviament havien estat sotmeses a estressos físics i fisicoquímics. Durant l’etapa final del projecte, els coneixements adquirits sobre la preparació de mostres per CF es van aplicar a l’anàlisi de mostres de matriu complexa: amanides comercials inoculades amb E. coli O157:H7. Als assajos amb indicadors d’integritat de membrana en suspensions bacterianes sotmeses a estrès, es van poder quantificar cèl•lules amb la membrana parcialment danyada (presumptes cèl•lules lesionades). El recompte de cèl•lules que mantingueren l’activitat respiratòria després de ser sotmeses a estrès va ser superior al que es va obtenir mitjançant recompte en placa convencional, cosa que va evidenciar la presència de cèl•lules actives però no cultivables. La introducció d’estratègies per reduir les interferències provocades per les partícules alimentàries i l’ús d’un anticòs amb marcatge fluorescent va permetre detectar selectivament les cèl•lules d’E. coli O157:H7 i avaluar-ne la integritat de membrana simultàniament. L’anàlisi de cèl•lules bacterianes per CF requereix de la exhaustiva optimització dels protocols, que són específics per cada soca i matriu. Malgrat això, i a diferència del mètode convencional per recompte en placa, ofereix la possibilitat d’obtenir una gran quantitat d’informació sobre el sovint complex estat fisiològic d’una mostra.

Diversity of the bacterial community in the surface soil of a pear orchard based on 16S rRNA gene analysis

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes was used to characterize bacterial populations in the surface soil of a commercial pear orchard consisting of different pear cultivars during two consecutive growing seasons. Pyrus communis L. cvs Blanquilla, Conference, and Williams are among the most widely cultivated cultivars in Europe and account for the majority of pear production in Northeastern Spain. To assess the heterogeneity of the community structure in response to environmental variables and tree phenology, bacterial populations were examined using PCR-denaturing gradient gel electrophoresis (DGGE) followed by cluster analysis of the 16S ribosomal DNA profiles by means of the unweighted pair group method with arithmetic means. Similarity analysis of the band patterns failed to identify characteristic fingerprints associated with the pear cultivars. Both environmentally and biologically based principal-component analyses showed that the microbial communities changed significantly throughout the year depending on temperature and, to a lesser extent, on tree phenology and rainfall. Prominent DGGE bands were excised and sequenced to gain insight into the identities of the predominant bacterial populations. Most DGGE band sequences were related to bacterial phyla, such as Bacteroidetes, Cyanobacteria, Acidobacteria, Proteobacteria, Nitrospirae, and Gemmatimonadetes, previously associated with typical agronomic crop environments

Multifunctionality and Diversity in Bacterial Biofilms

Bacteria are highly diverse and drive a bulk of ecosystem processes. Analysis of relationships between diversity and single specific ecosystem processes neglects the possibility that different species perform multiple functions at the same time. The degradation of dissolved organic carbon (DOC) followed by respiration is a key bacterial function that is modulated by the availability of DOC and the capability to produce extracellular enzymes. In freshwater ecosystems, biofilms are metabolic hotspots and major sites of DOC degradation. We manipulated the diversity of biofilm forming communities which were fed with DOC differing in availability. We characterized community composition using molecular fingerprinting (T-RFLP) and measured functioning as oxygen consumption rates, the conversion of DOC in the medium, bacterial abundance and the activities of five specific enzymes. Based on assays of the extracellular enzyme activity, we calculated how the likelihood of sustaining multiple functions was affected by reduced diversity. Carbon source and biofilm age were strong drivers of community functioning, and we demonstrate how the likelihood of sustaining multifunctionality decreases with decreasing diversity

Gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.