Tesis sobre el tema "Pseudomonas"

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2008.<br>Bibliography: p. 257-274.<br>Literature review -- General materials and methods -- Characterisation of strain collection -- Distribution of integrons and gene cassettes in pseudomonas -- Genomic context of pseudomonas integrons -- Evolutionary analysis of pseudomonas spp. integrons 199 -- Final discussion -- Appendix -- References.<br>Integrons associated with mobile genetic elements have played a central role in the emergence and spread of multiple antibiotic resistance in many pathogenic bacteria. However, the discovery of integrons in the chromosomes of diverse, non-pathogenic bacteria suggests that integrons have a broader role in bacterial evolution. The Pseudomonas stutzeri species complex is a well studied model for bacterial diversity. Members of the complex are genetically closely related, but sub-taxa are not able to be defined by exclusively shared sets of phenotypic characters. Rather, on the basis of total DNA:DNA similarity, Ps. stutzeri strains have been divided into 17 different groups (termed genomovars). Two Ps. stutzeri strains have been found to contain Chromosomal Integrons (CIs). This thesis involved exploration of the hypothesis that a CI was present in the common ancestor of the Ps. stutzeri species complex and assessed the impact of integrons on diversity across all Pseudomonads. The history and significance of integrons is discussed in Chapter 1 as part of a literature review, and general materials and methods are provided in Chapter 2. Chapters 3 - 6 comprise the sections in which data generated during my PhD project are presented. A comprehensive analysis of the relationships between the strains being analysed is presented in Chapter 3. In Chapter 4, results of PCR and hybridisation screening for integrons across the strain collection are presented. In Chapter 5 the recovery of additional integrons and in depth sequence analysis of the recovered integrons are described. Finally, Chapter 6 contains statistical analyses of integron-associated genes and Chapter 7 contains a final discussion the most significant findings. Twenty-three Pseudomonas spp. strains were screened for the presence of integrons. All but three were found to contain integron-like sequences; however, most integron sequences recovered contained inactivated core integrons. viii Despite having a chromosomal locus, integrons in Pseudomonas were found to have properties indicative of frequent horizontal transfer. Evidence was also obtained which suggests that integrons have been acquired at the same locus on multiple independent occasions. This has not been observed in other families of chromosomal integrons and suggests that the loci at which integrons in Pseudomonas are found are hotspots for recombination.<br>Mode of access: World Wide Web.<br>xiii, 274 p. ill

The stringent response (SR) is a global regulatory mechanism that allows bacteria to survive starvation. The plant surface is one environment where a fluctuation in nutrient availability is experienced. Because both Pseudomonas sp. DF41 and Pseudomonas chlororaphis PA23 are able to protect canola from the fungal pathogen Sclerotinia sclerotiorum when applied as a foliar spray, we sought to investigate the impact of this response on the antifungal activities of these two biocontrol strains. The SR exerts its effects on gene transcription through production of the alarmone(p)ppGpp. Metabolism of (p)ppGpp is governed by two enzymes; RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. To investigate how the SR affects the ability of strains DF41 and PA23 to inhibit the fungal pathogen,relA and relAspoT mutants were generated through allelic exchange. Strain DF41 relA and relAspoT mutants were found to exhibit increased antifungal activity due to enhanced lipopeptide (LP) antibiotic production. Addition of relA, but not spoT in trans restored the mutant phenotype to that of the parent. The influence of the SR on the regulatory mechanisms governing strain DF41 biocontrol was also investigated. It was determined that relA forms part of the Gac regulon while RpoS is under SR control. In fact, addition of rpoS in trans restored protease activity to wild-type levels, but did not attenuate antifungal activity. The SR mutants PA23relA and PA23relAspoT, also exhibited increased growth inhibition of S. sclerotiorum in vitro compared to the wild type. Both mutants showed enhanced production of the antifungal factors pyrrolnitrin, lipase and protease and were complemented by the addition of relA but not spoT. Herein, the SR was found to regulate that Gac system, QS, and RpoS. The presence of gacS or rpoS in multicopy restored the mutant phenotype to that of the wild type.In summary, these findings suggest that the SR negatively influences the biocontrol activities of strains DF41 and PA23. It is evident that the SR is merely one mechanism by which DF41 and PA23-mediated antagonism is regulated.

The opportunistic pathogen P. aeruginosa is a leading cause of hospital-accquired infections, and is also the primary cause of morbidity and mortality in patients with cystic fibrosis (CF). In this thesis, I describe the identification and characterization of a novel LysR-type transcription regulator (LTTR) of P. aeruginosa named BexR. I show that BexR exhibits reversible ON/OFF bistable expression, which leads to the bistable expression of several genes including one encoding a virulence factor. I present results suggesting that this bistable expression depends on positive feedback of BexR. This work illuminates the simplicity with which a transcription regulatory network can exhibit a complex behavior and generate phenotypic diversity in a clonal population.

The opportunistic human pathogen Pseudomonas aeruginosa controls virulence, production of secondary metabolites, motility, biofilm formation, growth in anaerobic conditions, intracellular and intercellular signalling and the switch from an acute to a chronic mode of infection at the transcriptional and post-transcriptional levels by modulation of the Gac/Rsm system. Cell density-dependent signal accumulation and environmental stimulators such as pH changes and ion limitation activate the GacS/GacA two-component system which in turn triggers transcription of the small regulatory RNAs RsmY and RsmZ. These sRNAs sequester multiple copies of the RNA-binding protein RsmA, antagonising its function. The RsmA/CsrA proteins act as translational repressors by binding to the GGA-motifs in the untranslated region of target mRNAs and blocking ribosome binding. In this study, the biological function of RsmN, an RsmA homologue with a conserved RNA-binding pocket but a distinct protein folding, the predicted autoregulatory mechanism of RsmN, the nature of target transcripts of RsmN, and the cross-regulation between the two Rsm proteins were investigated. The positive control of proteolytic and elastinolytic activities and swarming motility by RsmN has been demonstrated using single and inducible double deletion mutants of rsmN. Furthermore, rsmN deletion increased microcolony formation during biofilm formation. Regulation by RsmN was most apparent in the absence of RsmA, when rsmN expression was induced via a multicopy plasmid and at temperatures lower than 37°C. The double deletion of rsmA and rsmN affected growth, diminished proteolytic and elastinolytic activities, triggered autolysis and led to the increased secretion of the type VI secretion system protein Hcp1. Moreover, the double deletion of rsmA and rsmN altered the colony morphology of P. aeruginosa. Mutagenesis of the functionally critical, conserved RNA-binding residue which is identified as Arg44 in RsmA and Arg62 in RsmN resulted in the loss of RsmN function. In a genome-wide analysis by RNASeq, target transcripts were co-purified with RsmN from 37°C and 34°C cultures of a wild-type strain expressing rsmN in multicopy numbers. RNASeq results indicated that small regulatory RNAs such as CrcZ, RsmY and RgsA are common targets of RsmN and RsmA, whereas PhrS is a target of RsmN only. Other common RsmA and RsmN targets included transcriptional regulators, heat shock proteins, proteases, starvation response proteins, components of the denitrification pathway, outer membrane proteins required for pore formation, type III and type VI secretion system proteins and RsmA. Transcripts of heat shock proteins, the tss operon genes and rsmA were enriched by RsmN at 37°C but not at 34°C whereas the lasB transcript was enriched by RsmN at 34°C but not at 37°C. Based on the list of common targets of RsmA and RsmN and the results obtained from phenotypic assays, induction of the lytic Pf4 prophage, accumulation of alkyl quinolone or c-di-GMP signalling molecules, imbalanced redox state, carbon starvation, increased membrane permeability, and aggregation of misfolded proteins are suggested as possible mechanisms triggering the excessive autolysis of the rsmNind ΔrsmA mutant under uninducing conditions. The data gathered so far suggests that rsmN is differentially expressed, with increased RsmN activity at temperatures below 37°C in comparison with RsmA, and, RsmA and RsmN collectively contribute to the regulation of secondary metabolite production, motility and microcolony formation in P. aeruginosa.

The major outer membrane protein OprF from Pseudomonas aeruginosa was compared with OprF from the fluorescent phytopathogen Pseudomonas syringae. The P. syringae oprF gene was subcloned and sequenced and found to code for a sequence of 344 amino acids containing a 24 amino acid leader sequence. The mature protein, with a deduced molecular weight of 34,225, contained four cysteine residues and an alanine-proline rich area. Comparison of the P. syringae OprF amino acid sequence with the P. aeruginosa OprF and the E. coli OmpA sequences showed that the sequences were most similar at the carboxy-terrninal ends. Restriction enzyme site heterogeneity near the oprF gene from nine different P. syringae pathovars was determined. All pathovars had a conserved SalI site within the gene and conserved PstI. and BamHI sites near the ends of the gene. The location of the PstI and the SalI sites outside the gene was variable, although similar. Immunological relatedness between P. syringae OprF from the different pathovars and P. aeruginosa OprF was confirmed. Protein OprF from all the pathovars was shown to be 2-mercaptoethanol modifiable and more easily heat modifiable than was OprF from P. aeruginosa.<br>Science, Faculty of<br>Microbiology and Immunology, Department of<br>Graduate

La taxonomie des pseudomonas fluorescents phytopathogènes oxydase négative bien qu'ayant évolué dans le temps est actuellement floue et insatisfaisante. Ce groupe comprend 52 pathovars de p. Syringae et 5 espèces proches (p. Cichorii et p. Viridiflava, p. Amygdali, p. Ficuserectae, p. Meliae). Les caractères phénotypiques (20 caractères biochimiques et l'assimilation de 147 substrats hydrocarbones) de 655 souches des pathovars de p. Syringae et des 5 espèces proches ont été étudiés. Les données ont été interprétées par l'utilisation d'une méthode de taxonomie numérique et du coefficient de capacite de diagnostic (ccd). Les résultats font apparaitre la grande diversité phénotypique des pathovars p. Syringae. Des souches de certains pathovars comme pv. Glycineae, pv. Phaseolicola, pv. Savastanoi, pv. Porri, pv. Avellanae, pv. Persicae, pv. Tomato, pv. Morsprunorum, pseudomonas sp. (magnolia sp. ) Forment des phenons que l'on différencie par les caractères phénotypiques. D'autres pathovars comme pv. Syringae, pv. Pisi, pv. Aptata, pv. Atropurpurea, pv. Japonica, pv. Dysoxyli, pv. Eriobotryae, pv. Lapsa, pv. Striafaciens et pv. Atrofaciens s'agrègent dans le même phenon a des distances faibles et ne sont pas identifiables par les caractères biochimiques. Les caractères génomiques des souches-type des 52 pathovars de p. Syringae et des 5 espèces proches ont été étudiés par hybridations quantitatives adn/adn par la methode de la nucléase s1. Il a été mis en évidence 9 espèces génomiques qui regroupent un nombre variable de pathovars : p. Syringae (10), p. Savastanoi (20), p. Tomato (14), p. Porri (8), p. Avellanae (2), p. Helianthi (2), p. Tremae (1), p. Cannabinae (1) et p. Viridiflava (2). La validation de ces 9 espèces sera proposée. Cependant, aucune caractérisation biochimique ne permet pas encore leur identification.

Aspartate transcarbamoylase (ATCase) was purified from 16 selected bacterial species including existing Pseudomonas species and former species reassigned to new genera. An enormous diversity was seen among the 16 enzymes with each class of ATCase being represented. The smallest class, class C, with a catalytically active homotrimer, at 100 kDa, was found in Bacillus and other Gram positive bacteria. In this report, the ATCases from the Gram negatives, Shewanella putrefaciens and Stenotrophomonas maltophilia were added to class C membership. The enteric bacteria typify class B ATCases at 310 kDa, with a dodecameric structure composed of two catalytic trimers coupled to three regulatory dimers. A key feature of class B ATCases is the dissociability of the holoenzyme into regulatory and catalytic subunits which were enzymatically active. In this report, the ATCase from Pseudomonas indigofera was added to class B ATCases. The largest class, at 480 kDa, class A, contains the fluorescent Pseudomonas including most members of the 16S rRNA homology group I. Two polypeptides are produced from overlapping pyrBC' genes. The former, pyrB, encodes a 34 kDa catalytic polypeptide while pyrC' encodes a 45 kDa dihydroorotase-like polypeptide. Two non active trimers are made from six 34 kDa chains which are cemented by six 45 kDa chains to form the active dodecameric structure. Dissociation of the holoenyzme into its separate active subunits has not been possible. In this report, the ATCases from Comamonas acidovorans and C. testosteroni, were added to the class A enzymes. An even larger class of ATCase than class A at 600 kDa was discovered in Burkholderia cepacia. Stoichiometric measurements predict a dodecamer of six 39 kDa polypeptides and six 60 kDa polypeptides. Unlike other large pseudomonads ATCases, the enzyme from B. cepacia was dissociable into smaller active forms. Both the holoenzyme and its dissociated forms were regulated by nucleotide effectors. A new class of ATCase was proposed for B. cepacia type enzymes.

Pseudomonas corrugata and P. mediterranea, are two closely related bacteria both causal agents of tomato pith necrosis and recently evaluated as biocontrol agents and producers of industrially-promising biomolecules. The interest of our work focused on cyclic lipopeptides (CLPs) that are surface active molecules with antibacterial, antifungal and cytotoxic properties. P. corrugata is known to produce two kinds of cyclic lipopeptides, corpeptins and cormycin A. Investigations on P. corrugata strain CFBP5454 quorum sensing (QS) system helped to postulate its role on virulence mechanism mediated by CLPs during detrimental interaction with tomato plants. On the contrary no information was available on P. mediterranea/tomato molecular interaction. Both species are able to induce on tomato undistinguishable symptoms thus we thoughts that they likely share common virulence mechanisms. Starting from these premises we demonstrated that P. mediterranea CFBP 5447 harbours one system designated PmeI/R that is highly homologous to the PcoI/R system of P. corrugata and that virulence on tomatoes is affected by inactivation of QS genes in both species although with some differences. A LuxR regulator RfiA encoded by rfiA cotranscribed both with pcoI and pmeI was also demonstrated to be pivotal for virulence. The MALDI-TOF MS spectra of bacterial cell-free culture filtrates showed that P. corrugata CFBP5454 produces both corpeptins and cormycin and for the first time these CLPs were also found in a P. mediterranea strain which spectrum also showed a putatively unidentified bioactive compound. Mutational analysis revealed that P. mediterranea CFBP 5447 PmeI- and PmeR-, P. corrugata CFBP 5454 PcoR- and both RfiA-, were impaired in CLP production but also showed a drastic reduction of virulence when inoculated in tomato plants demonstrating their pivotal role. These results also evidence that the QS is involved in CLP production and all the correlated phenotypes (colony morphology, biosurfactant activity) via the RfiA transcriptional regulator. In the P. corrugata CFBP 5454 cosmid insert sequence from which the AHL QS genes were identified we describes three genes crpC, crpD, crpE. Bioinformatics and mutational analysis of crpC and crpDE demonstrated they are involved in the production and secretion of corpeptins since the derivative mutant strain cell-free culture filtrates showed only the peaks of cormycin in their MALDI-TOF MS spectra. Moreover, pathogenicity tests revealed that these long chain CLPs are important for P. corrugata virulence. Using real-time PCR we also ascertained that the regulators PcoR and RfiA are involved in corpeptins production/secretion. P. corrugata CFBP 5454 and P. mediterranea CFBP5447 produced in vitro diffusible compounds with antimicrobial properties which inhibited the growth of plant pathogenic fungi and bacteria, as well as antifungal volatile compounds. Antagonistic activity of QS mutants didn t different from that of their respective parent strains whereas antifungal activity was completely abolished in the rfiA- mutants. Since both the pcoR- (and pmeR-) and rfiA- mutants did not produce CLPs the production of one or more additional antimicrobial compound is suggested. In vivo experiment showed the good biocontrol activity of P. corrugata CFBP 5454 but also that the QS and rfiA mutants are still able to reduce the disease incidence in the in vivo investigated pathosystems. P. corrugata CFBP 5454 draft genome sequence helped in identifying a biosynthetic cluster hcnABC coding for the enzyme HCN synthase involved in this volatile compound biosynthesis. P. corrugata hcnA and hcnC knockout mutants failed to produce HCN and showed a reduced antimicrobial activity against Botrytis cinerea. Cyanogenesis seems to be not regulated either by AHL-QS or by RfiA because mutants still retained the ability to produce HCN.

Physical and restriction mapping of the salicylate catabolic plasmid SAL from Pseudomonas putida strain PpG 2119 was carried out by standard multiple restriction analysis and by cross hybridization studies using radioactively labeled restriction fragment probes. The total numbers of fragments produced, their respective sizes, the arrangement of the restriction fragments on the plasmid and the map locations of the enzyme recognition sites for Hpal, Xhol, Dral and Smal are given.

The interactions of macrophages with Pseudomonas aeruginosa were studied. Five monoclonal antibodies specific for porin protein F were tested for their ability to opsonize P. aeruginosa for complement-independent phagocytosis by unelicited mouse peritoneal macrophages, human peripheral blood monocytes and mouse macrophage cell line P388[sub D1]. All five antibodies significantly increased the level of bacterial uptake over that obtained with the non-opsonic controls. The relative effectiveness of the different antibodies was approximately the same in all cell types indicating that the P388[sub D1] cells can be used as a model for normal macrophages. Of the four monoclonal antibodies directed against similar epitopes of protein F, the three IgGl monoclonal antibodies were substantially more opsonic than the one IgG2a isotype. P. aeruginosa cytotoxin and periplasmic contents caused a significant reduction in antibody-mediated phagocytosis of P. aeruginosa. Phagocytosis was restored upon pre-incubation with anti-cytotoxin serum. Both cytotoxin and periplasmic contents caused depolarization of the P388[sub D1] cell membrane, as demonstrated using a polarization-sensitive fluorescent probe. These data indicated that P. aeruginosa cytotoxin was localized in the periplasm and had the potential to inhibit macrophage-mediated phagocytosis, possibly by perturbing ion gradients across the macrophage plasma membrane. Monoclonal antibodies directed against protein F were also capable of enhancing phagocytosis of in vivo-grown P. aeruginosa. P. aeruginosa cells taken directly from the in vivo growth system were significantly more susceptible to macrophage phagocytosis than were the same cells after being washed in buffer. The phagocytosis-promoting factor could be isolated from the supernatant of in vivo-grown bacteria and was determined to be fibronectin. Data indicated that promotion by fibronectin of non-opsonic phagocytosis was mediated by direct activation of the macrophages. The tetrapeptide arginine-glycine-aspartate-serine in the eukaryotic cell binding domain of fibronectin was demonstrated to be the macrophage-activating region. Phagocytosis of a mutant P. aeruginosa strain lacking surface pili could not be enhanced by fibronectin. Furthermore, exogenously added Pseudomonas pili was capable of abrogating the enhanced phagocytosis of the wild type strain observed with fibronectin-activated macrophages. It was concluded that Pseudomonas pili were the bacterial ligands required for attachment to fibronectin-activated macrophages in the initial stages of non-opsonic phagocytosis.<br>Science, Faculty of<br>Microbiology and Immunology, Department of<br>Graduate

This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.

Orientador: Avelino Rodrigues de Oliveira<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia<br>Made available in DSpace on 2018-07-13T22:59:50Z (GMT). No. of bitstreams: 1 Benedetti_CelsoEduardo_M.pdf: 3663805 bytes, checksum: 5f4bbe356452eca96dba02a8c2f8ff04 (MD5) Previous issue date: 1991<br>Resumo: Culturas de Pseudomonas avenae (MANNS, 1909) e Pseudomonas rubrilineans (LEE et aI, 1925) STAPP, 1928] , provenientes de diferentes regiões e oriundas de milho, teosinte, arroz e cana-de-açucar, foram comparadas através da análise eletrotorética de proteínas totais e de membrana, em gel de poliacrilamida-SDS (EGPA-SDS) . Testes para verificar a sensibilidade das culturas a antibióticos e avaliar a produção de bacteriocinas também foram realizados visando a comparação dos isolados. A análise eletroforética das proteínas da fração de membrana revelou diferenças nos perfis de proteínas de baixo peso molecular, entre 24.000 e 14.000. O agrupamento das culturas bacterianas feito apenas com base nos perfis de proteínas de baixo peso molecular foi semelhante ao agrupamento obtido através da análise numérica dos densitogramas desses perfis. Através desses dados verificou-se uma possível relação entre perfis de proteínas e hospedeira de origem das culturas bacterianas. Através da análise eletroforética das proteínas totais da bactéria, não foi possível a diferenciação dos isolados de P. avenae e P. rubrilineans. Além disso, as diferenças observadas nos padrões de bandas de baixo peso molecular das proteínas de membrana não são observadas nos perfis de proteínas totais. Culturas de P. avenae e P. rubrilineans também não diferiram quanto à sensibilidade aos antibióticos ampicilina, tetraciclina, streptomicina, cloranfenicol e kanamicina, em diferentes concentrações. O teste de produção de bacteriocina mostrou culturas de P. avenae produturas e culturas de P. rubrilineans indicadoras dessa substância. Os resultados obtidos nesse trabalho sugerem sinonímia entre culturas de P. avenae e P. rubrilineans. Entretanto, a análise das frações de proteínas de membrana mostram grupos de isolados distintos originários de milho e cana-de-açúcar, como também, algumas culturas de P. avenae e P. rubrilineans apresentam características distintas, tanto no padrão de proteínas de membrana, como na produção de bacteriocina ou em testes de patogenicidade. Discutiu-se a necessidade ainda do emprego de outros critérios de análise taxonômica no estudo dessas bactérias, tais como a análise de fragmentos de restrição do DNA<br>Abstract: Electrophoretical analysis (SDS-PAGE) of whole cell proteins and membrane fraction proteins were used to compare Pseudomonas avenae (MANNS, 1909) with Pseudomonas rubrilineans (LEE et al, 1925) STAPP, 1928) strains from different hosts. Antibiotic sensitivity and bacteriocin production tests were also analised. The electrophoretical analysis of the membrane -fraction protein showed differences in low molecular weight protein profiles, between MW 24.000 and 14.000. Strains which were grouped by numerical analysis of protein profiles were similar to those based only on low molecular weight -protein profiles. It was verified a possible relationship between protein profiles and the original host of strains. No differences were observed between P. avenae and P. rubrilineans strains when the whole cell protein samples were compared. Moreover, the differences observed in low molecular weight proteins of the-membrane fractions were not found in total protein profiles. P. avenae and P. rubrilineans did not differ on sensit ivity to ampicilin, tetracycline, streptomycin, chloramphenicol and kanamycin, in different concentrations. The results of this work suggest sinonymy of P. avenae and P. rubrilineans cultures, however, the analysis of membrane proteins have shown distinct strain groups from corn and sugarcane. Also, some P. avenae and P. rubrilineans strains showed distinct characteristics in bacteriocin production and host assay tests. The use of other techniques, such as restriction DNA profile, to improve the understanding of the taxonomic position of these bacteria is discussed.<br>Mestrado<br>Biologia Vegetal<br>Mestre em Ciências Biológicas

La large distribution des Pseudomonas fluorescens est liée à leur grande adaptabilité aux facteurs de stress tels que les variations environnementales. Ce travail avait pour objet la réponse spécifique au milieu aéroporté de P. Fluorescens, comme l’aéroportée MFAF76a et la clinique MFN1032 comme standard. La technique récente HPTLC-MALDI-TOF MSI a permis de caractériser les divers glycérophospholipides (GP) des deux souches. En phase stationnaire de croissance, un GP inconnu (UGP - unknown GP) a été isolé et semble intervenir dans l’adaptation à la température de la souche clinique MFN1032. Quant au stress NO2 gaz, les deux souches ont été exposées aux concentrations: 0. 1, 5 et 45 ppm. Leurs phénotypes ont été confrontés à l’expression de quelques gènes ciblés. Pour les valeurs standard 0,5 and 5 ppm en NO2, aucun paramètre n’est modifié. Par contre, une réponse bactérienne est constatée suite à l’exposition à 45 ppm de NO2. Cette exposition semble impacter la production d’UGP. De plus hmp et mexEF-oprN, codant respectivement pour la flavohémoglobine et pour la pompe à efflux RND, se trouvent surexprimés, corroborant l’évolution de la résistance bactérienne aux antibiotiques. Contrairement au NO, aucune altération de la biomasse de biofilm n’est observée pour le NO2, qui favorise cependant l’augmentation de son épaisseur chez MFAF76a, mais aussi l’inhibition du swarming et la diminution du swimming, avec un taux de c-di-GMP stable. Ce faisceau de résultats offre, pour la première fois, la réponse bactérienne et le rôle des GP lors de stress comme NO2 ou à la température, autre modification environnementale<br>The high distribution of Pseudomonas fluorescens group is linked to its ability to adapt to stress factors. This work goaled the response of an airborne P. Fluorescens MFAF76a, and its clinical standard P. Fluorescens MFN1032 to environmental changes in order to refine the specific adaptation of airborne bacteria. First the HPTLC-MALDI TOF MSI tool defined glycerophospholipid (GP) composition of both strains. In stationary growth phase, an unknown GP, in short UGP, was found and seemed to be involved in temperature adaptation for the clinical strain. After exposure to 0. 1, 5 and 45 ppm concentrations, the bacterial response to NO2 was defined through motility, biofilm formation, antibiotic resistance and expression of several chosen target genes. While no change in parameters was seen in bacteria exposed to 0. 1 and 5 ppm of NO2, several alterations were occurred with a bacterial exposure to 45 ppm. NO2 seemed to bias the UGP production, reduced P. Fluorescens swim and decreased swarm only for MFN1032 strain. Biofilm formed by NO2-treated MFAF76a showed increased maximum thickness, with no change in c-di-GMP intracellular level. Expression of the hmp-homologue gene involved in NO detoxification was upregulated in response to NO2, suggesting a possible common pathway between NO and NO2 detoxification. Finally, NO2 was found to increase bacterial resistance to ciprofloxacin and chloramphenicol. Thus the resistance nodulation cell division (RND) MexEF-OprN efflux pump encoding genes were highly upregulated in both strains. Together these findings implement the first model of bacterial response to NO2 toxicity and the role(s) of GP in bacterial adaptation to environmental changes

Orientador: Marcelo Cristianini<br>Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos<br>Made available in DSpace on 2018-08-03T20:56:17Z (GMT). No. of bitstreams: 1 Senatore_MarcelaAndreaDuranHaun_M.pdf: 684290 bytes, checksum: 522970fe79261c7d2890c0a19a39b587 (MD5) Previous issue date: 2004<br>Resumo: Um dos problemas que enfrenta a indústria de laticínios é a recontaminação do leite decorrente da formação de biofilmes em tanques de armazenamento e trocadores de calor. A tecnologia de esterilização de materiais por gás plasma tem sido utilizada com sucesso na esterilização de instrumentos cirúrgicos, oftálmicos e dentários. Os objetivos deste trabalho foram avaliar a eficiência de um sistema de esterilização a gás plasma sob células de Pseudomonas fluorescens aderidas em placas de aço inoxidável utilizando o leite como substrato. Foram avaliadas as variáveis tempo de pré plasma, tempo de exposição ao plasma e potência do mesmo na remoção do biofilme. As placas de aço inoxidável com a bactéria aderida foram submetidas a um tratamento com gás plasma, que foi formado a partir de um produto comercial composto por ácido peracético, peróxido de hidrogênio e ácido acético. Dois sistemas modelo foram utilizados para simular a formação de biofilmes de Pseudomonas fluorescens, um dinâmico e outro estático, simulando trocadores de calor e tanques de armazenamento, respectivamente. Através do modelo estático foi possível obter contagens acima de 105 UFC por placa de aço inoxidável, mesmo após três ciclos de lavagem das placas em água estéril sob agitação. Foi observado um efeito positivo na inativação de P. fluorescens em função do tempo de pré-plasma do sanificante. Exposições acima de 7 minutos foram capazes de produzir reduções superiores a dois ciclos logarítmicos na inativação do microrganismo. Através de um planejamento experimental fatorial 23 foi demosntrado que as variáveis tempo de pré-plasma, tempo de exposição ao plasma e potência do plasma apresentaram efeitos positivos na inativação de Pseudomonas fluorescens. O tempo de exposição (min.) apresentou o maior efeito na destruição da bactéria; mas sendo um pouco superior à potência do plasma (w)<br>Abstract: One of the problems that food industry is facing is the milk recontamination through biofims formation on storage tanks and heat exchanger. The technology of sterilization for materials through gas plasma has been used with succesfull for cirurgycal, ophathalmic and dentistry equipment. The goals of this work was to evaluate the efficiency of a gas plasma sterilization system on Pseudomonas fluorescens cells adhered on stainless steel plates using milk as substrate. Time of pre plasma, plasma exposition and potency (Watts) were evaluated as independent variables on cell destruction. The stainless steel plates were submitted to gas plasma treatment originated from a commercial product composed of peracetic acid, hydrogen peroxide and acetic acid. Two model systems were used to simulate a biofilm formation of Pseudomonas fluorescens, one dynamic and one static, in order to simulate heat exchangers and storage tanks, respectively. Through static model it was possible to get counts over 105 UFC/plate after washing three times using sterile water under stirring conditions. It was observed a positive effect on P. fluorescens inactivation by pre-plasma in a time dependent way. Expositions over seven minutes were capable to produce reductions higher than two logarithmic cycles on microorganism inactivation. A 23 factorial design indicated that the pre-plasma time, time exposition and potency showed positive effects on Pseudomonas fluorescens inactivation. Time exposition (min) was the most effective variable on bacteria destruction, being a little higher than plasma potency (w)<br>Mestrado<br>Mestre em Tecnologia de Alimentos

A 1.57 kb XhoI restriction fragment derived from the TOL plasmid pDKI was subcloned into the E. Coli plasmid pUC19. The complete nucleotide sequence of this XhoI fragment was determined using both the chemical cleavage and chain termination DNA sequencing methods.

Pseudomonas aeruginosa est une bactérie pathogène opportuniste qui peut développer deux modes d’infection. Les infections aigües sont associées à la production et à la sécrétion de toxines qui peuvent avoir un effet cytotoxique, alors que dans le contexte d’une infection chronique la bactérie a tendance à s’établir sous la forme d’un biofilm. Un biofilm est une population de microorganismes organisée en une communauté et attachée sur une surface. Cette surface peut être biotique ou abiotique. Les biofilms bactériens ont des caractéristiques intrinsèques qui les rendent plus résistants à des conditions environnementales difficiles (pH, oxygène, UV, flux, etc…). Dans le cadre d’une infection l’établissement du biofilm résulte aussi en une population bactérienne difficile à éliminer par le système immunitaire mais également résistante aux antibiotiques. Des déterminants moléculaires majeurs qui interviennent dans la formation du biofilm sont le flagelle, les pili de type IV et les fimbriae Cup pour l’attachement, ou bien encore les exopolysaccharides qui sont avec l’ADN des composants essentiels de la matrice extracellulaire du biofilm qui englobe la population bactérienne. Chez P. aeuginosa, si l’alginate est le polysaccharide majeur, il a été montré que des souches non-mucoides forment également des biofilms et que dans ce contexte la synthèse et la sécrétion du polysaccharide majeur de la matrice sont dépendantes des gènes pel. Mon travail a consisté dans l’étude de l’organisation structurale du système Pel et le contrôle de son activité<br>Pseudomonas aeruginosa is a human opportunistic pathogen, and in the course of an infection can develop two lifestyles. One of them is involved in secretion of toxins and results in acute infection, and the other one causes chronic infections and is characterized by formation of a biofilm. A biofilm is a highly organized bacterial population, organized as a complex community that is attached to a surface. Bacterial biofilm shows higher resistance to different enviromental factors including physical factors, antimicrobials or host immune response. Major components taking part in biofilm formation are flagella, type IV pili, cup fimbriae as well as exopolysaccharides. The latter provides a protective matrix for the biofilm, together with proteins and DNA. In non-mucoid strains the major exopolysaccharide of biofilm matrix is synthetised and secreted by pel gene cluster. My work concentrates on the structural organisation of Pel polysaccharide secretion machinery, regulation of its activity as well as detailed characterisation of the Pel polysaccharide itself

The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation. As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The physiological relevance of this is explained.

El principal objetivo de la Tesis Doctoral es el estudio en profundidad de poblaciones de Pseudomonas presentes en el ambiente. Para ello se ha realizado una prospección de los aislamientos de Pseudomonas procedentes de diversos hábitats. Se han analizado muestras en suelos agrícolas, arenas de la zona intermareal, aguas freáticas y aguas de ríos. Los aislados de Pseudomonas aeruginosa procedentes de muestras ambientales, se han comparado con los aislados clínicos procedentes del Hospital Universitario Son Dureta. Se han aplicado distintas metodologías en el análisis de estos estudios. Métodos dependientes de cultivo, basados en metodología tradicional, y métodos independientes de cultivo basados en nuevos métodos moleculares. Estos últimos incluyen: tipado de secuencias multilocus, secuenciación con cebadores específicos y análisis de DNA total procedente de muestras ambientales por clonación y pirosecuenciación con cebadores específicos. Esta última metodología se ha aplicado por primera vez en muestras ambientales de la manera realizada en esta Tesis. La genómica comparativa se ha aplicado para evaluar la diversidad intraclonal. Se ha estimado el potencial de nuevas metodologías moleculares como la clonación, pirosecuenciación, y estudio de genomas, aplicándolo al análisis de la diversidad de las especies de Pseudomonas. Los datos obtenidos nos permiten tener una amplia perspectiva de estos métodos aplicados a la ecología y taxonomía del género Pseudomonas. En el primer capítulo, se ha analizado la estructura y microdiversidad de 53 aislamientos de muestras ambientales y clínicas de Mallorca (España), mediante el análisis de secuencias multilocus (MLST). Patrones de multiresistencia a distintos antibióticos, solo se hallaron en aislamientos de origen clínico. El elevado número de nuevos alelos y secuencias tipo, halladas en una misma área, refleja la gran diversidad de las poblaciones de P. aeruginosa. A todo ello deben sumarse los índices de diversidad que también indicaron la alta diversidad de la población estudiada. Los tests de clonalidad demostraron que la recombinación juega un papel esencial en la distribución de los alelos. La secuencia tipo ST-1146 fué la única secuencia hallada en los dos tipos de muestras, tres aislamientos en muestras ambientales y un aislado clínico con distinto perfil de resistencia a antibióticos. En el segundo capítulo, los cuatro genomas de los aislados ST-1146 fueron secuenciados y ensamblados de novo. Los resultados indicaron que el número de genes propios del aislado clínico (SD9) era superior al de los aislados de origen ambiental (P37, P47 y P49). Genes relacionados con el bacteriófago Pf1 y otros relacionados con los bacteriófagos F116 y H66 solo se hallaron en SD9 pero no en las otras cepas del ST-1146 de origen ambiental. Los genes relacionados con el bacteriófago Pf1 de SD9 presentaron un elevado número de mutaciones respecto a los aislados de origen ambiental. La comparación genómica demostró que los aislados ST-1146 están estrechamente relacionados y los genes relacionados con la patogenicidad estudiados estaban conservados. El número de alelos exclusivos de SD9 fue 2,5 y 3,6 veces superior a los aislados de origen ambiental, al compararse todos ellos con los genomas de referencia de las cepas P. aeruginosa PAO1-UW y UCBPP-PA14. En el tercer capítulo, el río Woluwe se tomó como hábitat modelo para el estudio de la diversidad de las especies del género Pseudomonas. Una muestra de agua no contaminada se analizó por métodos dependientes e independientes de cultivo. La identificación de los aislados de Pseudomonas se analizó por secuenciación y análisis de los cebadores del gen rpoD. Los métodos independientes de cultivo se basaron en la clonación y pirosecuenciación del amplicón del gen rpoD obtenido con los cebadores selectivos para dicho gen en Pseudomonas: PsEG30F-PsEG790R. Cabe destacar el elevado número de cepas de Pseudomonas obtenidas en las muestras por los tres métodos de análisis: 26 especies distribuidas en 13 grupos o subgrupos filogenéticos. La pirosecuenciación ha sido el mejor método de los utilizados; las secuencias obtenidas correspondieron a 24 de las especies totales observadas, con la excepción de P. stutzeri y P. simiae. El grupo filogenético predominante fue Pseudomonas fluorescens. En todos los métodos de análisis se halló un gran número de posibles nuevas especies indicando una enorme diversidad del género, no descrita hasta el momento. En el cuarto capítulo, se aislaron cepas de Pseudomonas de muestras ambientales procedentes de suelos y zonas intermareales. En el proceso de identificación algunos de estos aislamientos no han podido ser asignados a especies conocidas de Pseudomonas considerándose como posibles nuevas especies. En el análisis de secuencias multilocus se incluyeron cepas procedentes de nuestra colección del laboratorio de Microbiología de la “Universitat de les Illes Baleares”. El análisis de secuencias multilocus demostró que varios aislados podrían corresponder a 3 nuevas especies (6, 5 y 1 aislados de cada especie). La confirmación de estos resultados requerirá posteriores análisis. Otros cuatro aislados fueron estudiados mediante su caracterización morfológica, fisiológica, bioquímica, quimiotaxonómica y genómica. Estos estudios demostraron que los aislados no podían ser asignados a ninguna especie conocida de Pseudomonas por lo que se han propuesto dos cepas nuevas: Pseudomonas aestusnigri (cepa VGXO14T = CECT 8317 T = CCUG 64165 T) y Pseudomonas terricola (cepa S58 T = CECT 8389 T = CCUG 64415 T).<br>The main goal of the present work is a thorough study of the diversity of Pseudomonas populations present in several habitats. In chapter one, the population structure and microdiversity of 53 Pseudomonas aeruginosa isolates from environmental samples and clinical specimens obtained in Mallorca (Spain) has been analyzed by a multilocus sequence typing approach (MLST). Antibiotic multiresistance to several antibiotics was only found in isolates of clinical origin. The high number of new alleles and new sequence types found in a limited area reflects the great diversity of P. aeruginosa populations and the high plasticity of a paradoxically phylogenetic conserved genome of P. aeruginosa. The calculated genetic diversity index also demonstrated the high diversity of the population under study. Clonality tests demonstrated that recombination plays a key role in the distribution of alleles. The ST-1146 was the only one found in both kind of samples, 3 environmental isolates (from the same site isolated at 2 different dates) and 1 clinical isolate, with differences in its antibiotic susceptibility profile. For this reason, the 4 genomes of newly described sequence type ST-1146 have been sequenced and analyzed. In the second chapter, the four genomes of ST-1146 were obtained and the sequences assembled de novo and compared with the CD-HIT program. Results showed that the number of isolate-specific genes was higher in the clinical isolate (SD9) than in environmental isolates (P37, P47 and P49). Some genes related to phage Pf1 and to other phages similar to bacteriophages F116 and H66 were found in isolate SD9 but not in the other isolates of ST-1146. The bacteriophage Pf1 region in isolate SD9 accumulated the highest number of mutations in comparison with the environmental isolates. Comparative genomic methods indicated that the isolates of ST-1146 are closely related, and most genes implicated in pathogenicity are highly conserved in the environmental isolates, suggesting the genetic potential for infectivity similar to that of the clinical isolate. Moreover, the four genomes were mapped against the reference genomes of P. aeruginosa PAO1-UW and UCBPP-PA14. A mutational profile was performed as a result of each comparison. The clinical isolate showed in both comparisons a number of exclusive alleles 2.5 and 3.6 times greater than the environmental isolates. These results suggest that the mutation pressure is not the same in the environmental isolates than in the clinical one. In the third chapter, the River Woluwe has been taken as a model habitat for the study of the diversity of species in the genus Pseudomonas. A water sample from a non-contaminated site at the source of the river was analyzed by culture-dependent and –independent methods. Identification of the Pseudomonas isolates was performed by sequencing and analysis of their rpoD sequence. Culture-independent methods consisted of a cloning and pyrosequencing of a specific rpoD amplicon obtained from total DNA extracted from the same sample and amplified by Pseudomonas rpoD primer set EGPsF340-EGPsR 780. It was remarkable the number of known species detected in the sample by the three different methods: 26 species distributed in 13 phylogenetic groups or subgroups within the genus. Pyrosequencing was the more powerful analysis; sequences obtained represented the 24 species with the exception of P. stutzeri and P. simiae. The predominant phylogenetic group within the Pseudomonas genus was Pseudomonas fluorescens group in the cultures and in the culture-independent analysis. In all analysis a high number of putative novel species were found indicating the enormous diversity not described yet. In the fourth chapter, several Pseudomonas strains have been isolated from environmental samples, from soil and intertidal habitats. In the identification process, some of these strains have not been assigned to known Pseudomonas species and were considered members of putative novel species. In their phylogenetic characterization by MLSA we found that strains in the culture collection of our laboratory were close-related and therefore they were also included in the taxonomic characterization of these putative novel species. MLSA demonstrated that 3 putative novel species were represented by 6, 5 and 1 strains respectively, which will be the subject of additional studies. Four other strains were deeply studied by a taxonomic polyphasic approach, including morphological, physiological, biochemical, chemotaxonomic and genomic characterizations. These studies demonstrated that the four strains cannot be assigned to any of the known Pseudomonas species and we propose the creation of two novel species, Pseudomonas aestusnigri (strain VGXO14T = CECT 8317 T = CCUG 64165 T) and Pseudomonas terricola (strain S58 T = CECT 8389 T = CCUG 64415 T).

Pseudomonas fluorescens SBW25 (P.f.SBW25) is a plant growth-promoting rhizobacterium (PGPR) that efficiently colonises the rhizosphere of a range of plants. Previously In Vivo Expression Technology (IVET) has identified a rhizosphereinduced gene (pinA) in P.f.SBW25 that shows homology to members of the nitrilase group of hydrolyzing enzymes. Nitrilase enzymes catalyse the hydrolysis of nitrile ;J) compounds (R-CN) to the corresponding carboxylic acid and ammonia. These enzymes have been described in plants, animals, bacteria and fungi and may be important in hormone synthesis, detoxification of toxic nitriles and nitrogen acquisition. In this investigation· nitrilase activity has been examined in P.f.SBW25 and in a range of other plant-associated Pseudomonas to identify the substrates and potential roles of nitrilase enzymes in bacteria colonising the plant environment. ~-cyanoalanine (AlaCN), a nitrile produced by plants and bacteria during cyanide detoxification, acts as a inducing chemical and a substrate for pinA activity. pinA expression is also induced by the substrates for AlaCN synthesis, cyanide and cysteine. Heterologous expression of pinA in bacteria and plants confers AlaCN hydrolysing activity and increased tolerance to toxic levels of AlaCN, suggesting a role for pinA in AlaCN detoxification. AlaCN-hydrolysing activity has also been observed in Pseudomonas f1uorescens PfO-1 and Pf-5. Nitrilase activity has also been detected in the plant pathogenic bacterium Pseudomonas syringae pv. syringae B728a (P.s.B728a). This bacterium hydrolyses arylacetonitriles, such as indoleacetonitrile, which are abundant in the plant environment and may do so for the purposes of nitrogen utilisation, detoxification or to modify plant growth and development.

The pathogenic bacterium Pseudomonas aeruginosa uses a variety of highly efficient chelating compounds (siderophores) to acquire sufficient iron for growth and virulence. These siderophores can either be endogenous or acquired from exogenous sources such as other bacteria or fungi. The transport of the endogenous siderophore pyoverdine activates a signal-transduction pathway that increases the synthesis of both the ferripyoverdine receptor protein (FpvA) and pyoverdine itself. Signal-transduction systems similar to this have three specific proteins involved: a receptor protein specific for one siderophore in the outer membrane, an anti-sigma factor in the cytoplasmic membrane and a sigma factor that activates gene expression in the cytoplasm. The aim of the research presented in this thesis was to study the roles of the proteins in three different iron uptake and signalling pathways of P. aeruginosa. The substrates for each receptor protein were confirmed and the roles of each protein in the pathways were compared to the P. aeruginosa pyoverdine signalling pathway. The pyoverdine, desferrioxamine and ferrichrome transport pathways were studied to find whether interactions occur between them and if so, the mechanism(s) for that interaction. Furthermore, a technique for analysing gene expression of P. aeruginosa in sputum from the cystic fibrosis (CF) lung was developed. This technique was subsequently used to study the levels of iron responsive gene expression. The receptor, sigma factor and anti-sigma factors were all found to have a role in the siderophore-induced expression of their own signalling pathway. The experimental data provide evidence of similarities in the roles of the sigma and receptor proteins within each pathway but different roles for the anti-sigma factors. In the absence of the cognate sigma factor or anti-sigma factor the expression of the desferrioxamine and ferrichrome receptors could not be upregulated. Without its cognate sigma factor fpvA could no longer be upregulated in the presence of pyoverdine. However, unlike the other systems, in the absence of the cognate anti-sigma factor, expression of fpvA was always observed. This is consistent with the anti-sigma factors being required for the activity of the cognate sigma factor in the ferrichrome and desferrioxamine signalling pathways but not the pyoverdine signalling pathway. The siderophore signalling pathways were found to be upregulated in the presence of multiple siderophores, but generally to a lesser extent than if only one siderophore was available. This suggests that in the presence of multiple siderophores, P. aeruginosa uses all available iron chelators. The study of the role of the receptor, sigma factor and anti-sigma factor into these effects indicate sigma factor competition for RNA polymerase has a major role in the effects of multiple siderophores on pathways upregulation. The gene expression studies of P. aeruginosa in sputum from the lungs of CF patients provided support for the hypothesis that the bacteria were growing in an environment where iron levels were sufficient for bacterial growth, but not storage of iron. The expression of the sigma factor gene pvdS that is required for pyoverdine synthesis was studied because expression of this gene is a sensitive reporter of intracellular iron levels. It was found to be downregulated in bacteria in sputum compared to laboratory grown bacteria. This result suggests the bacteria are inhabiting a more iron-replete environment within the lung. This finding advances our understanding of the CF lung environment and the impact it has on P. aeruginosa infection. This knowledge has medical implications for the development of novel therapies to combat P. aeruginosa infection.

The characteristics of the effects of chloramphenicol on Pseudomonas aeruginosa were examined. Resistant strains were easily isolated following a single passage in chloramphenicol at 150 $ mu$g/ml to 500 $ mu$g/ml. Drug detoxification or altered sensitivity of the target site could not be the mechanism of resistance. This resistance to chloramphenicol was correlated with the addition of an outer membrane protein with a molecular weight of 49 kDa and the loss of two outer membrane proteins, one with the molecular weight of 19 kDa and the other of about 10 kDa. The highly specific requirement of the resistant strains for Ca$ sp{2+}$, Mg$ sp{2+}$, Mn$ sp{2+}$ or Sr$ sp{2+}$ described by Irvin and Ingram (1982) was confirmed by the observation that the outer membrane of the resistant cells contained twice as much Mg$ sp{2+}$ cation as the sensitive cells. Many other experiments designed to observe the effects of chloramphenicol on the outer membrane of P. aeruginosa failed. It was concluded that the observations made in this study strongly suggested a "re-structuring" of the outer membrane of P. aeruginosa, rendering the resistant cells more impermeable to chloramphenicol.

<i>Pseudomonas tolaasii</i> is the causal agent of brown blotch disease of mushrooms. The bacterium exists in two stable phenotypic forms: the smooth, virulent wild type (1116S), which produces the toxin tolaasin; and the rough, avirulent variant (1116R), which does not produce tolaasin. The PheN-PheS two-component signal transduction pathway regulates the phenotype of these forms. Spontaneous phenotypic switching between the 1116S and 1116R is mediated by a reversible 661 bp duplication within the sensor region of the <i>pheN</i> gene. The role of the environment in phenotypic switching between 1116S and 1116R was studied. It was found that frequency of switching from 1116R to 1116S in colonies was enhanced with the addition of mushroom extract to the medium. Characterisation of the active component of mushroom demonstrated the signal to be heat labile, active at 1,000 fold dilution and <12,000 kDa in size. Further characterisation suggested the presence of 2 distinct signal molecules in the mushroom extract, one in the cationic fraction and one in an anionic fraction. The toxin, tolaasin, a major determinant of phenotype is produced in a cell density dependent manner. The role of <i>N</i>-acyl homoserine lactone (AHL) mediated quorum sensing regulation of phenotype was studied. Thin layer chromatography indicated that 1116S produces the autoinducer compound, <i>N -</i>(3-oxododecanoyl)-L-homoserine lactone (OdDHL), while 1116R does not produce a detectable level of AHLs, suggesting that AHL production is under the regulation of the PheN-PheS two-component signal transduction pathway. Further investigation demonstrated the presence of an unidentified secondary quorum sensing autoinducer, which is only present below an OD₆₀₀ of 1.0. The addition of synthetic OdDHL to culture medium prior to inoculation with 1116S did not induce tolaasin synthesis, suggesting that either OdDHL is not involved with the regulation of tolaasin, or that a secondary level of regulation also exists.