A Holistic Approach from Systems Biology Reveals the Direct Influence of the Quorum-Sensing Phenomenon on Pseudomonas aeruginosa Metabolism to Pyoverdine Biosynthesis.

Computational modeling and simulation of biological systems have become valuable tools for understanding and predicting cellular performance and phenotype generation. This work aimed to construct, model, and dynamically simulate the virulence factor pyoverdine (PVD) biosynthesis in Pseudomonas aeruginosa through a systemic approach, considering that the metabolic pathway of PVD synthesis is regulated by the quorum-sensing (QS) phenomenon. The methodology comprised three main stages: (i) Construction, modeling, and validation of the QS gene regulatory network that controls PVD synthesis in P. aeruginosa strain PAO1; (ii) construction, curating, and modeling of the metabolic network of P. aeruginosa using the flux balance analysis (FBA) approach; (iii) integration and modeling of these two networks into an integrative model using the dynamic flux balance analysis (DFBA) approximation, followed, finally, by an in vitro validation of the integrated model for PVD synthesis in P. aeruginosa as a function of QS signaling. The QS gene network, constructed using the standard System Biology Markup Language, comprised 114 chemical species and 103 reactions and was modeled as a deterministic system following the kinetic based on mass action law. This model showed that the higher the bacterial growth, the higher the extracellular concentration of QS signal molecules, thus emulating the natural behavior of P. aeruginosa PAO1. The P. aeruginosa metabolic network model was constructed based on the iMO1056 model, the P. aeruginosa PAO1 strain genomic annotation, and the metabolic pathway of PVD synthesis. The metabolic network model included the PVD synthesis, transport, exchange reactions, and the QS signal molecules. This metabolic network model was curated and then modeled under the FBA approximation, using biomass maximization as the objective function (optimization problem, a term borrowed from the engineering field). Next, chemical reactions shared by both network models were chosen to combine them into an integrative model. To this end, the fluxes of these reactions, obtained from the QS network model, were fixed in the metabolic network model as constraints of the optimization problem using the DFBA approximation. Finally, simulations of the integrative model (CCBM1146, comprising 1123 reactions and 880 metabolites) were run using the DFBA approximation to get (i) the flux profile for each reaction, (ii) the bacterial growth profile, (iii) the biomass profile, and (iv) the concentration profiles of metabolites of interest such as glucose, PVD, and QS signal molecules. The CCBM1146 model showed that the QS phenomenon directly influences the P. aeruginosa metabolism to PVD biosynthesis as a function of the change in QS signal intensity. The CCBM1146 model made it possible to characterize and explain the complex and emergent behavior generated by the interactions between the two networks, which would have been impossible to do by studying each system's individual components or scales separately. This work is the first in silico report of an integrative model comprising the QS gene regulatory network and the metabolic network of P. aeruginosa.

BIOFILMS AND EXTRACTS FROM BACTERIA PRODUCING "QUORUM SENSING' SIGNALING MOLECULES PROMOTE CHEMOTAXIS AND SETTLEMENT BEHAVIORS IN Hydractinia Symbiolongicarpus (Cnidaria: Hydrozoa) LARVAE / Biopelículas y extractos de bacterias que producen moléculas de señalización de "Quorum Sensing' promueven comportamientos de quimiotaxis y asentamiento en larvas de Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa)

ABSTRACT Many sessile marine invertebrates have life cycles involving the development of larvae that settle on specific substrates to initiate metamorphosis to juvenile forms. Although is recognized that bacterial biofilms play a role in this process, the responsible chemical cues are beginning to be investigated. Here, we tested the role of substrate-specific bacteria biofilms and their Quorum Sensing Signaling Molecule (QSSM) extracts on chemotaxis and settlement of larvae from Hydractinia symbiolongicarpus, a hydroid that grows on gastropod shells occupied by hermit crabs. We isolated and taxonomically identified by 16S rDNA sequencing, 14 bacterial strains from shells having H. symbiolongicarpus. Three isolates, Shigella flexneri, Microbacterium liquefaciens, and Kocuria erythromyxa, were identified to produce QSSMs using biosensors detecting N-acyl-L-homoserine lactones. Multispecies biofilms and QSSM extracts from these bacteria showed a positive chemotactic effect on H. symbiolongicarpus larvae, a phenomenon not observed with mutant strains of E. coli and Chromobacterium violaceum that are unable to produce QSSMs. These biofilms and QSSMs extracts induced high rates of larval attachment, although only 1 % of the attached larvae metamorphosed to primary polyps, in contrast to 99 % of larvae incubated with CsCl, an artificial inductor of attachment and metamorphosis. These observations suggest that bacterial QSSMs participate in H. symbiolongicarpus substrate selection by inducing larval chemotaxis and attachment. Furthermore, they support the notion that settlement in cnidarians is decoupled into two processes, attachment to the substrate and metamorphosis to a primary polyp, where QSSMs likely participate in the former but not in the latter.

RESUMEN Muchos invertebrados marinos sésiles tienen ciclos de vida que involucran el desarrollo de larvas que se asientan en sustratos específicos iniciando su metamorfosis a formas juveniles. Aunque es conocido que biopelículas bacterianas participan en este proceso, las señales químicas responsables hasta ahora se empiezan a investigar. Aquí evaluamos el papel de biofilms bacterianos y sus extractos de moléculas de señalización de "Quorum Sensing' (QSSM) sobre la quimiotaxis y el asentamiento larvario en Hydractinia symbiolongicarpus, un hidrozoario que crece sobre conchas de gastrópodos ocupadas por cangrejos ermitaños. Nosotros aislamos e identificamos taxonómicamente por secuenciación de rDNA 16S 14 cepas bacterianas de conchas con H. symbiolongicarpus. Tres de ellas, Shigella flexneri, Microbacterium liquefaciens, and Kocuria erythromyxa, mostraron producción de QSSMs usando biosensores que detectan N-acil-L-homoserin lactonas. Biopelículas y extractos de QSSMs de estas bacterias mostraron efectos quimiotácticos sobre larvas de H. symbiolongicarpus, efecto no observado en ensayos con cepas mutantes de E. coli y Chromobacterium violaceum que son incapaces de producir QSSMs. Las biopelículas y sus extractos indujeron adhesión larvaria sobre superficies, aunque solamente el 1 % de las larvas asentadas hicieron metamorfosis hacia pólipo primario, en contraste con 99 % de larvas incubadas con CsCl, un inductor artificial de asentamiento y metamorfosis. Estas observaciones sugieren que QSSMs de biopelículas bacterianas participan en la selección de sustrato de H. symbiolongicarpus, induciendo quimiotaxis y asentamiento de sus larvas. También sugieren que el asentamiento en cnidarios tiene dos procesos, adhesión y metamorfosis, donde las QSSMs participarían en el primero, pero no en el segundo.

A synthetic multi-cellular network of coupled self-sustained oscillators.

Engineering artificial networks from modular components is a major challenge in synthetic biology. In the past years, single units, such as switches and oscillators, were successfully constructed and implemented. The effective integration of these parts into functional artificial self-regulated networks is currently on the verge of breakthrough. Here, we describe the design of a modular higher-order synthetic genetic network assembled from two independent self-sustained synthetic units: repressilators coupled via a modified quorum-sensing circuit. The isolated communication circuit and the network of coupled oscillators were analysed in mathematical modelling and experimental approaches. We monitored clustering of cells in groups of various sizes. Within each cluster of cells, cells oscillate synchronously, whereas the theoretical modelling predicts complete synchronization of the whole cellular population to be obtained approximately after 30 days. Our data suggest that self-regulated synchronization in biological systems can occur through an intermediate, long term clustering phase. The proposed artificial multicellular network provides a system framework for exploring how a given network generates a specific behaviour.

Biofilm inhibition activity of compounds isolated from two Eunicea species collected at the Caribbean Sea

Abstract Biofilm has a primary role in the pathogenesis of diseases and in the attachment of multicellular organisms to a fouled surface. Because of that, the control of bacterial biofilms has been identified as an important target. In the present study, five lipid compounds isolated from soft coral Eunicea sp. and three terpenoids together with a mixture of sterols from Eunicea fusca collected at the Colombian Caribbean Sea showed different effectiveness against biofilm formation by three marine bacteria associated with immersed fouled surfaces, Ochrobactrum pseudogringnonense,Alteromona macleodii and Vibrio harveyi, and against two known biofilm forming bacteria, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923. The pure compounds were characterized by NMR, HRESI-MS, HRGC-MS and optical rotation. The most effective compounds were batyl alcohol (1) and fuscoside E peracetate (6), acting against four strains without affecting their microbial growth. Compound 1 showed biofilm inhibition greater than 30% against A. macleodii, and up to 60% against O. pseudogringnonense,V. harveyi and S. aureus. Compound 6 inhibited O. pseudogringnonense and V. harveyi between 25 and 50%, and P. aeruginosa or S. aureus up to 60% at 0.5 mg/ml. The results suggest that these compounds exhibit specific biofilm inhibition with lower antimicrobial effect against the bacterial species assayed.

Possible ecological role of pseudopterosins G and P-U and seco-pseudopterosins J and K from the gorgonian Pseudopterogorgia elisabethae from Providencia Island (SW Caribbean) in regulating microbial surface communities.

The gorgonian Pseudopterogorgia elisabethae collected at Providencia Island (Colombia) has an unfouled surface, free of obvious algal and invertebrate growth. This gorgonian produces significant amounts of the glycosilated diterpenes pseudopterosins and seco-pseudopterosins (Ps and seco-Ps). Our previous experiments have shown activity of these compounds against eukaryotic (human cancer cell lines and Candida albicans) and prokaryotic cells (Staphylococcus aureus and Enterococcus faecalis). However, the potential role of pseudopterosins on the regulation of the fouling process is still under study. We evaluated the activity of these compounds against bacteria isolated from heavily fouled marine surfaces as an indicator of antifouling activity. Additionally, we assessed their activity against bacteria isolated from P. elisabethae to determine whether potentially they play a role in preventing surface bacterial colonization, thus impairing presumptively the establishment of further successional stages of fouling communities. Results showed that Ps and seco-Ps seem to modulate bacterial growth (controlling Gram-positive bacterial growth and inducing Gram-negative bacterial associations). We thus hypothesized that Ps and seco-Ps may play a role in controlling microbial fouling communities on the surface of this gorgonian. By using bTEFAP and FISH we showed that the most abundant bacteria present in the microbial communities associated with P. elisabethae are Gram-negative bacteria, with Proteobacteria and Gammaproteobacteria the most representative. To evaluate whether Ps and seco-Ps have a direct effect on the structure of the bacterial community associated with P. elisabethae, we tested these compounds against culturable bacteria associated with the surface of P. elisabethae, finding remarkable selectivity against Gram-positive bacteria. The evidence presented here suggests that Ps and seco-Ps might have a role in the selection of organisms associated with the gorgonian surface and in the regulation of the associated bacterial community composition.

Disruption in quorum-sensing systems and bacterial biofilm inhibition by cembranoid diterpenes isolated from the octocoral Eunicea knighti.

Three new cembranoid diterpenes, knightine (1), 11(R)-hydroxy-12(20)-en-knightal (2), and 11(R)-hydroxy-12(20)-en-knightol acetate (3), were isolated as minor constituents of the Caribbean gorgonian Eunicea knighti, along with the known cembranoids 4-8. The stereostructures of the new compounds were determined by detailed spectroscopic analyses and a combination of chemical transformations and modified Mosher's methods. All isolated cembranoids were tested against fouling using a quorum-sensing inhibition (QSI) assay and a biofilm inhibition test. Compounds 2, 3, and 6 disrupted QS systems at lower concentrations than kojic acid and Cu(2)O, and in most cases cembranoids 1-8 showed bacterial biofilm inhibition at lower concentrations than kojic acid.

Shifts in bacterial communities of two Caribbean reef-building coral species affected by white plague disease.

Coral reefs are deteriorating at an alarming rate mainly as a consequence of the emergence of coral diseases. The white plague disease (WPD) is the most prevalent coral disease in the southwestern Caribbean, affecting dozens of coral species. However, the identification of a single causal agent has proved problematic. This suggests more complex etiological scenarios involving alterations in the dynamic interaction between environmental factors, the coral immune system and the symbiotic microbial communities. Here we compare the microbiome of healthy and WPD-affected corals from the two reef-building species Diploria strigosa and Siderastrea siderea collected at the Tayrona National Park in the Caribbean of Colombia. Microbiomes were analyzed by combining culture-dependent methods and pyrosequencing of 16S ribosomal DNA (rDNA) V5-V6 hypervariable regions. A total of 20,410 classifiable 16S rDNA sequences reads were obtained including all samples. No significant differences in operational taxonomic unit diversity were found between healthy and affected tissues; however, a significant increase of Alphaproteobacteria and a concomitant decrease in the Beta- and Gammaproteobacteria was observed in WPD-affected corals of both species. Significant shifts were also observed in the orders Rhizobiales, Caulobacteriales, Burkholderiales, Rhodobacterales, Aleteromonadales and Xanthomonadales, although they were not consistent between the two coral species. These shifts in the microbiome structure of WPD-affected corals suggest a loss of community-mediated growth control mechanisms on bacterial populations specific for each holobiont system.

Antifouling activities against colonizer marine bacteria of extracts from marine invertebrates collected in the Colombian Caribbean Sea and on the Brazilian coast (Santa Catarina).

The growth inhibition of 12 native marine bacteria isolated from Aplysina sponge surfaces, the shell of a bivalve, and Phytagel immersed for 48 h in sea water were used as indicator of the antifouling activity of the extracts of 39 marine organisms (octocorals, sponges, algae, and zoanthid) collected in the Colombian Caribbean Sea and on the Brazilian coast (Santa Catarina). Gram-negative bacteria represented 75% of the isolates; identified strains belonged to Oceanobacillus iheyensis, Ochrobactrum pseudogrignonense, Vibrio campbellii, Vibrio harveyi, and Bacillus megaterium species and seven strains were classified at genus level by the 16S rRNA sequencing method. The extracts of the octocorals Pseudopterogorgia elisabethae, four Eunicea octocorals, and the sponges Topsentia ophiraphidites, Agelas citrina, Neopetrosia carbonaria, Monanchora arbuscula, Cliona tenuis, Iotrochota imminuta, and Ptilocaulis walpersii were the most active, thus suggesting those species as antifoulant producers. This is the first study of natural antifoulants from marine organisms collected on the Colombian and Brazilian coasts.

Antiherpes screening of marine organisms from Colombian Caribbean Sea

The exploration of marine environment represents a promising strategy in the search for new active antiviral compounds. The isolation and characterization of the nucleosides spongothymidine and spongouridine from the sponge Cryptotethia crypta used as models for the synthesis of ara-A (vidarabine), that has been used therapeutically against herpetic encephalitis, was the most important contribution since the late 1970s. This paper describes the in vitro antiviral evaluation of 26 organic extracts obtained from eleven octocoral species and fifteen marine sponges. Cytotoxicity was evaluated on Vero cells by MTT assay and the antiviral activity was tested against Herpes Simplex Virus type 1 (HSV-1, KOS strain) by plaque number reduction assay. Results were expressed as 50 percent cytotoxic (CC50) and 50 percent inhibitory (IC50) concentrations, respectively, in order to calculate the selectivity index (SI= CC50/IC50) of each extract. Among the tested marine octocoral species, only three extracts showed antiviral activity, but with low selectivity indices (<3.0). Among the tested marine sponges, eight extracts showed SI values higher than 2.0, and three can be considered promising (Aka cachacrouense, Niphates erecta and Dragmacidon reticulatum) with SI values of 5.0, 8.0 and 11.7, respectively, meriting complementary studies in order to identify the bioactive components of these sponge extracts, which are in course now.

La proteomica n la era postgenómica / The Proteomics In The Postgenomic Era

El principal desafío de la biología moderna es entender la expresión, función y regulación del conjunto completo de proteínas codificadas por un organismo, lo cual describe el objetivo del nuevo campo de la proteómica. Las proteínas son las efectoras del trabajo celular, por ello el estudio de sus perfiles globales de expresión y de sus cambios bajo determinadas condiciones fisiológicas o patológicas, permite entender la red compleja de interacciones en que se basa el funcionamiento de una célula. La electroforesis en dos dimensiones (2D-PAGE) es la técnica central de la proteómica. En la actualidad no existe otro método con la capacidad para resolver simultáneamente miles de proteínas en un solo procedimiento y para detectar modificaciones post y co-traduccionales imposibles de predecir a partir de la secuencia genómica. Sus aplicaciones incluyen el análisis de proteomas, señalización, detección de marcadores de enfermedades y cáncer.

The main challenge of modern biology is to understand the expression, function and regulation of the whole set of proteins codified by an organism, which is the objective of the new field of proteomics. Proteins are the effectors of cellular work and the knowledge of their global expression profiles and changes under physiological and pathological conditions can help us to understand the complex network of interactions involved in cellular function. Two-dimensional electrophoresis (2-DE) is the central technology in proteomics. At present no other technique has the throughput and high resolution of 2-DE for the separation of thousands of proteins in one procedure and for the analysis of post-and co-translation modifications, not predictable from the genome sequence. The scope of applications extends from proteome analysis, to cell signaling, disease markers and cancer.

Cembranoid diterpenes from the Caribbean sea whip Eunicea knighti.

Three new cembranoid diterpenes, knightol (1), knightol acetate (2), and knightal (3), along with the known asperdiol (4) and asperdiol acetate (5), were isolated as major compounds from the sea whip Eunicea knighti collected from the Colombian Caribbean. The structures and absolute configurations of 1-5 were determined on the basis of spectroscopic analyses and by a combination of chemical and NMR methods, multiple correlations observed in a ROESY experiment, and using the modified Mosher method. Additionally, five semisynthetic compounds, 6-10, obtained during the chemical transformations of the natural compounds are here reported for the first time. All compounds were tested for antimicrobial activity against marine bacteria associated with heavily fouled surfaces and were also screened for antiquorum sensing (QS) activity. Compounds 1, 3, and 8 showed significant antimicrobial activity against bacterial isolates, and 1, 3, 7, and 8 showed excellent anti-QS inhibition activity measured by means of bioluminescence inhibition with biosensor model systems.

Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics.

Pseudomonas putida strains are frequently isolated from the rhizosphere of plants and many strains promote plant-growth, exhibit antagonistic activities against plant pathogens and have the capacity to degrade pollutants. Factors that appear to contribute to the rhizosphere fitness are the ability of the organism to form biofilms and the utilization of cell-to-cell-communication systems (quorum sensing, QS) to co-ordinate the expression of certain phenotypes in a cell density dependent manner. Recently, the ppu QS locus of the tomato rhizosphere isolate P. putida Iso F was characterized and an isogenic QS-negative ppuI mutant P. putida F117 was generated. In the present study we investigated the impact of QS and biofilm formation on the protein profile of surface-associated proteins of P. putida IsoF. This was accomplished by comparative proteome analyses of the P. putida wild type IsoF and the QS-deficient mutant F117 grown either in planktonic cultures or in 60 h old mature biofilms. Differentially expressed proteins were identified by peptide mass fingerprinting and database search in the completed P. putida KT2440 genome sequence. The sessile life style affected 129 out of 496 surface proteins, suggesting that a significant fraction of the bacterial genome is involved in biofilm physiology. In surface-attached cells 53 out of 484 protein spots were controlled by the QS system, emphasizing its importance as global regulator of gene expression in P. putida IsoF. Most interestingly, the impact of QS was dependent on whether cells were grown on a surface or in suspension; about 50% of the QS-controlled proteins identified in planktonic cultures were found to be oppositely regulated when the cells were grown as biofilms. Fifty-seven percent of all identified surface-controlled proteins were also regulated by the ppu QS system. In conclusion, our data provide strong evidence that the set of QS-regulated proteins overlaps substantially with the set of proteins differentially expressed in sessile cells.

Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone isolates.

Two strains, Pseudomonas aeruginosa TB10839 and TB121838, which belong to the TB clonal lineage, have been isolated from sputa of cystic fibrosis patients. Despite the fact that the strains are closely related, their pathogenic potential differs dramatically: while strain TB10839 is capable of proliferating in polymorphonuclear granulocytes, strain TB121838 is not. Comparative two-dimensional polyacrylamide gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry was employed to map the extracellular, intracellular, and surface sub-proteomes of TB10839 and TB121838 and to identify differentially expressed proteins. About 4% of all detected protein spots were differentially expressed between both strains including absent or present spots and spots with a more than 2-fold changed intensity. This percentage reflects a relatively high degree of intraclonal variability. Many of the protein spots in TB10839 that were missing or expressed at lower levels in TB121838 were identified as quorum-sensing regulated virulence factors. It might be speculated that the increased expression of these proteins contributes to pathogenic competence of TB10839.

Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics.

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N-acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem.

Analysis of the quorum-sensing regulon of the opportunistic pathogen Burkholderia cepacia H111 by proteomics.

Burkholderia cepacia H111, an important pathogen for persons suffering from cystic fibrosis, employs a quorum-sensing (QS) system, cep, to control expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are only expressed when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. In this study, we compared the protein pattern of the intracellular, extracellular, and surface protein fractions of an AHL-deficient cepI mutant with the one of the parent strain H111 by means of two-dimensional gel electrophoresis (2-DE). Our analysis showed that 55 proteins out of 985 detected spots were differentially expressed; these are expected to represent QS-controlled gene products. Addition of the respective signal molecules to the growth medium of the cep mutant fully restored the wild-type protein expression profile. In total about 5% of the B. cepacia proteome was downregulated and 1% upregulated in the cepI mutant, indicating that quorum sensing represents a global regulatory system. Nineteen proteins were identified with high confidence by N-terminal sequence analysis.