Cooperation is a trait that is found at all levels of biological organization. Interestingly, cooperation appears to occur in bacteria that produce small, easily diffusible molecules called autoinducers. To understand why bacteria produce these autoinducers, the scientific community has focused on one predominant theory called quorum sensing. Under this theory, bacteria produce autoinducers so they can sense the density of the population. Once a sufficiently high population density is reached, autoinducers initiate the production of a costly gene product that serves to benefit the population. In contrast, a competing theory called diffusion sensing suggests that autoinducers are used by the individual cells and are not used for cooperation. Here, the production of the autoinducer serves as a mechanism to sense environmental conditions. If the environmental conditions are favorable, a costly gene product is produced. To what extent, and under what conditions, are each of these opposing theories valid remains to be identified. In this thesis, an engineered strain of Escherichia coli was used to identify the conditions under which quorum sensing and diffusing sensing can be observed. It was discovered that, depending upon the frequency at which the spatial distribution of the autoinducer and bacteria was disrupted, the population of engineered bacteria displayed hallmarks of either quorum sensing or diffusion sensing. Specifically, when the spatial distribution was disturbed at high or low frequency, quorum sensing was observed. However, when spatial distribution was disturbed at an intermediate frequency, diffusion sensing was observed. Understanding how these disturbances affect survival in bacteria may result in novel treatments for bacterial infections. In more general applications, it may be exploited in the development of alternative mechanisms for controlling invasive species or aid in species reintroduction.

The black band disease (BBD) microbial consortium often causes mortality of reef-building corals. Microbial chemical interactions (i.e., quorum sensing (QS) and antimicrobial production) may be involved in the BBD disease process. Culture filtrates (CFs) from over 150 bacterial isolates from BBD and the surface mucopolysaccharide layer (SML) of healthy and diseased corals were screened for acyl homoserine lactone (AHL) and Autoinducer-2 (AI-2) QS signals using bacterial reporter strains. AHLs were detected in all BBD mat samples and nine CFs. More than half of the CFs (~55%) tested positive for AI-2. Approximately 27% of growth challenges conducted among 19 isolates showed significant growth inhibition. These findings demonstrate that QS is actively occurring within the BBD microbial mat and that culturable bacteria from BBD and the coral SML are able to produce QS signals and antimicrobial compounds. This is the first study to identify AHL production in association with active coral disease.

This thesis examined the antimicrobial activity of select neotropical plants from Costa Rica and traditional Q’eqchi Maya medicines from Belize. In particular the potential for interference with bacterial quorum sensing (QS) and biofilm formation as well as fungal growth were assessed. Of one hundred and twenty six extracts collected from Costa Rica, one third showed significant QS inhibition while 13 species displayed more biofilm inhibitory activities than the positive control allicin. The active species belonged to the Lepidobotryaceae, Melastomataceae, Meliaceae, Sapindaceae, and Simaroubaceae. Twelve Marcgraviaceae species were tested for the same biological activities; of these, three showed similar QS inhibition to that of the positive control Delisea pulchra (Greville) Montagne and five with at least 30% biofilm inhibition. Only one species inhibited fungal growth – Marcgravia nervosa Triana & Planch. Bioassay-guided isolation of this plant resulted in the identification of the active principle as a naphthoquinone, with a minimum inhibitory concentration (MIC) ranging from 85 to 100 μM against Saccharomyces cerevisiae. Similarly, sixty one Q’eqchi’ Maya medicinal plant species were evaluated for their antimicrobial activities. Of these, four species showed more QS inhibition than D. pulchra, seven with comparable biofilm inhibitory activities that of allicin, and two with similarly antifungal activity to berberine. Two spirostanol saponins were isolated from Cestrum schlechtendahlii G.Don, an active antifungal plant. The major saponin showed growth inhibition against Saccharomyces cerevisiae and Fusarium graminearum, with MICs of 16.5 μM and 132 μM, respectively. Further analyses of this compound using chemical genomics suggested that its antifungal mechanism of action is pleiotropic, affecting multiple targets. Taken together, these findings showed that neotropical plants and traditional Q’eqchi’ Maya medicines contain phytochemicals that interfere with bacterial biofilm formation and quorum sensing as well as fungal growth.

For many years, researchers have studied the social lives of bacteria to understand intra- and inter-species interactions. Cell-cell communication, also known as quorum sensing (QS), is used by bacteria to coordinate their behaviour in response to environmental conditions. The QS system in Streptococcus species is well known to regulate competence. Studies show that Streptococcus pneumoniae has two homologous QS systems: 1) the competence (Com) system that regulates competence; and 2) a bacteriocin-like peptide (Blp) system that regulates the production of bacteriocins. Both functions are widespread in the genus. In S. pneumoniae, the Blp QS system shares a common ancestor and has similar features to the Com QS system. However, the evolutionary relationship between these QS systems remains obscure. SUCRE methodology was developed to identify the QS homologous genes in the streptococcal species. SUCRE uses four complementary approaches: homology search, putative gene finding, regulon construction, and evolutionary analysis. The performance of SUCRE was assessed in comparison with other orthology detection methods. SUCRE is precise in identifying the QS homologous genes and has similar performance to OrthoMCL. The QS system structures are found to be conserved across the streptococcal species. A streptococcal species phylogeny was constructed from the ribosomal and tRNA synthetase gene families. Using the QS genes identified from SUCRE and the streptococcal species phylogeny, the study infers the evolution of the QS systems in Streptococcus species. The study shows that the QS systems evolved as a regulon unit. The paralogous relationship between each of the QS systems suggests that duplication has a huge influence on functional divergence of the QS systems in the genus. Although, horizontal gene transfer (HGT) is commonly found in bacteria, little evidence is found to support that the effect of HGT on the functional divergence of the QS systems in this genus. However, the QS regulon genes of the same QS system are found to be non- vertically transferred across species that signifies that the HGT event promotes the sequence variation between these genes.

Pseudomonas aeruginosa é uma gamaproteobactéria com capacidade de colonizar diversos tipos de ambiente e infectar hospedeiros filogeneticamente distintos. Em humanos, comporta-se como um patógeno oportunista,estando frequentemente relacionada à infecções em indivíduos imunocomprometidos e indivíduos portadores de fibrose cística. Um mecanismo importante para a versatilidade de P. aeruginosa é o sistema de percepção de quórum (QS), onde a bactéria pode vincular expressão gênica à densidade populacional e às características do ambiente. Atualmente, sabe-se que muitos outros reguladores estão interligados com QS, entre eles, a proteína reguladora RsmA e os pequenos RNAs RsmZ e RsmY. Além disso, diversos fatores importantes para a patogenicidade da bactéria são reguladas por QS. Em P. aeruginosa PA14, um fator importante para a patogenicidade em diversos hospedeiros é a proteína KerV, cujo envolvimento com QS foi descrito pela primeira vez neste trabalho. A linhagem D12, que possui uma deleção no gene kerV, mostrou alterações em fenótipos regulados por QS, como a maior produção de piocianina, composto que contribui para virulência e persistência das infecções causada por P. aeruginosa. Por ser facilmente detectável e pela regulação de sua síntese não ter sido completamente explorada em PA14, a expressão dos genes responsáveis pela produção de piocianina é um interessante repórter na investigação do possível envolvimento de KerV com QS. Além de piocianina, D12 apresenta níveis reduzidos de ramnolipídeos. Esses fenótipos somados se assemelham aos fenótipos da mutação de rsmA, sugerindo o envolvimento de KerV com os sistemas QS e Gac-Rsm direta ou indiretamente. Neste trabalho, mostramos que KerV exerce um efeito negativo na regulação dos operons phz1 e phz2, responsáveis pela síntese de piocianina, alterando a expressão desses genes. KerV exerce também um efeito positivo na expressão da proteína RsmA, responsável pela repressão de diversos genes alvos, onde RsmA se liga ao sítio de ligação ao ribossomo no mRNA, impedindo a tradução. Ensaios de gel shift mostraram que a ligação direta de RsmA na sequência líder de phzA1 e phzA2 ocorre, elucidando a maneira pela qual KerV está envolvido na regulação da expressão dos operons phz em P. aeruginosa PA14. Mostramos também que phz2 é ativo e contribui para a síntese de piocianina, pois na ausência de phz1, os níveis do pigmento são maiores do que aqueles detectados em PA14. Isso sugere uma maior expressão de phz2 e uma regulação diferencial dos operons de acordo com as condições ambientais como possível estratégia para manter os níveis desse composto. Uma evidência dessa regulação diferencial é vista no mutante lasR. Na fase inicial de crescimento, esse mutante não produz piocianina, porém quando exposto a tempos mais longos de cultivo, a produção de piocianina é maior quando comparada a PA14. Isso é reflexo da ativação da expressão de phz1 no mutante lasR em fase estacionária tardia, enquanto phz2 permanece não expresso. Isso indica que phz2 é dependente de LasR, ainda que indiretamente. Já phz1, embora tenha sua expressão influenciada por LasR no estágio inicial de crescimento, na fase estacionária é regulado por outros fatores independentes de las. / Pseudomonas aeruginosa is a gammaproteobacterium that colonizes several environments and infects phylogenetically distinct hosts. It behaves as an opportunistic pathogen in humans, often related to infection in immunocompromised individuals and cystic fibrosis patients. An important mechanism for P. aeruginosa versatility is the quorum sensing (QS) network, that allows bacteria to link gene expression to population density and environmental traits. Several additional regulators are interconnected with QS, as the regulatory mRNA binding protein RsmA and the non-coding small RNAs RsmZ and RsmY. Futhermore, key factors for pathogenicity are QS-regulated. In P. aeruginosa PA14, an important pathogenicity-related factor is the KerV protein, described for the first time here as involved in QS. D12 strain, that harbor a deletion in the kerV gene, shows alterations in QS-regulated phenotypes, such as high production of pyocyanin, a compound that contributes to virulence and persistence of P. aeruginosa infections. As the production of pyocyanin is easily detected and all mechanisms involved in its synthesis regulation are not fully described, the expression of genes responsible for production of this pigment is a good reporter to investigate KerV involvement in the QS network. Additionally, D12 also shows lower levels of rhamnolipids, another QS-regulated trait. Taken together, these phenotypes resemble the effects of a rsmA mutation, suggesting KerV involvement with QS and Gac-Rsm systems. In this work, we propose that KerV exerts a negative effect in the regulation of phz1 and phz2 operons, responsible for pyocyanin synthesis, by alterating the expression of these genes. KerV also has a positive effect on rsmA expression, responsible for the repression of several genes by blocking the ribosome binding site preventing the translation. Gel shift assays showed that RsmA binds directly in the leader sequence of phzA1 and phzA2, elucidating the manner in which KerV is involved in the regulation of phz operons expression in P. aeruginosa PA14. We also demonstrate that phz2 is actively expressed and contributes to pyocyanin production in PA14, since in the phz1 mutant the levels of pyocyanin are even higher than in the wild type strain. This suggests a phz2 higher expression and a differential regulation of phz operons according to environmental changes as a mechanism to maintain the levels of pyocyanin synthesis. An evidence for this regulation is the synthesis of pyocyanin by the lasR mutant, which does not make pyocyanin at early growth stages. However, at late stationary phase, pyocyanin production is even higher than in the wild-type strain, reflecting the LasR-independent regulation of phz1 expression, while phz2 operon remains silent.

Quorum sensing is a mechanism used by many proteobacteria to regulate expression of target genes in a population-dependent manner. The quorum sensing system of Vibrio fischeri activates the luminescence (lux) operon when the autoinducer signaling molecule (N-3-oxohexanoyl homoserine lactone) is recognized and bound by the activator protein LuxR. LuxR subsequently binds to the lux box centered at à 42.5 bp upstream of the transcription initiation site and activates transcription from the lux operon promoter, resulting in the emission of light at high cell densities. LuxR consists of 250 amino acids arranged into an N-terminal (regulatory) domain and a C-terminal (activation) domain, and is thought to function as an ambidextrous activator capable of making multiple contacts with the alpha and sigma subunits of RNA polymerase (RNAP). Published work describing the results of alanine scanning mutagenesis performed on the C-terminal domain of LuxR (residues 190-250) has identified residues (K198, W201 and I206) that appear to play a role in positive control of transcription initiation. Additional mutagenesis of residues 180-189 has been undertaken via a three-primer or four-primer PCR-based method in this study. Variants of LuxR were screened for their ability to activate luciferase production and to repress transcription from an artificial promoter, and production of full-length LuxR was measured, in an attempt to identify additional positive control variants. No additional positive control variants were found in this study. Work has also been undertaken to identify intergenic suppressors between positive control variants of LuxR and the RNAP alpha subunit, RpoA. Starting with a recombinant Escherichia coli strain encoding the lux operon and LuxR variant I206E, a random chemical mutagenesis was performed on a vector encoding RpoA. Following transformation of the mutated plasmids encoding RpoA, high throughput luminescence assays were used to identify isolates with phenotypes brighter than the control. Isolation of an intergenic suppressor will confirm the existence of protein-protein interactions between LuxR and RpoA within the transcription initiation complex. The ability of other LuxR family members to establish productive protein-protein interactions with RNAP necessary for transcription initiation was also examined. LuxR homologues EsaR of Pantoea stewarti ssp. stewartii, a repressor of known function, and ExpR of Erwinia carotovora subsp. carotovora were also analyzed for their ability to activate the lux operon, as well as to repress transcription from an artificial promoter containing the lux box. / Master of Science

Collective robotic behavior poses significant advantages over classical control methods such as system response and robustness. Biological cooperative communities have provided great insights for development of many control algorithms. Localized chemical signaling within bacterial communities is used for directed movement and dynamic density measurements. Both individual and population scale models have been created to adequately model community dynamics. These dynamics, including directed motion due to chemotaxis and density controlled functionality from quorum sensing, are modeled through an individual scale in a community scale environment. This modeling provides both a platform for analyzing the BacteriaBot engineered system as well as inspires decentralized stochastic control techniques for solving bacteria-like collaborative control problems. / Master of Science

No description available.

The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.Two mutant transporters, SSU1 and CDR4, were characterized for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. While homoserine lactone showed a decrease in biofilm density of both mutants compared to the wild-type strain, wild-type and ssu1 biofilm densities increased considerably in the presence of cyclic-di-GMP while testing lower inocula. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.We determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. Our results suggest these two genes are downstream targets in a pathway regulated by Chk1p.

Bio-inspired swarming behaviors provide an effective decentralized way of coordinating robot teams. However, as robot swarms increase in size, bandwidth and time constraints limit the number of agents a human can communicate with and control. To facilitate scalable human interaction with large robot swarms it is desirable to monitor and influence the collective behavior of the entire swarm through limited interactions with a small subset of agents. However, it is also desirable to avoid situations where a small number of agent failures can adversely affect the collective behavior of the swarm. We present a bio-inspired model of swarming that exhibits distinct collective behaviors and affords limited human interaction to estimate and influence these collective behaviors. Using a simple naive Bayes classifier, we show that the global behavior of a swarm can be detected with high accuracy by sampling local information from a small number of agents. We also show that adding a bio-inspired form of quorum sensing to a swarm increases the scalability of human-swarm interactions and also provides an adjustable threshold on the swarm's vulnerability to agent failures.