<p>U okviru ove doktorske disertacije prikazan je postupak izolacije i karakterizacije denitrifikatora uz optimizaciju biotehnolo&scaron;kih uslova proizvodnje biomase i ispitivanje efikasnosti proizvedene biomase u pogledu bioči&scaron;ćenja različitih građevinskih materijala.<br />U okviru istraživanja koja su obuhvaćena ovom doktorskom disertacijom, izolovan je nov soj denitrifikatora koji je pomoću metode MALDI TOF identifikovan kao Pseudomonas stutzeri. U svim eksperimentima karakteristike i performanse novoizolovanog soja P. stutzeri, interno označenog kao D1, su poređene sa referentnom kulturom P. stutzeri ATCC 17588, koji je model mikroorganizam na kome se izučava proces denitrifikacije. Primenom PCR metode utvrđeno je postojanje četiri ključna denitrifikujuća gena (napA, nirS, norB i nosZ) u genomu P. stutzeri D1 i referentnog soja, čime je pokazano da ispitivani sojevi imaju genetsku predispoziciju za izvođenje potpune aerobne denitrifikacije. Takođe, uz primenu Boks-Benkenovog eksperimentalnog dizajna i metode odzivne povr&scaron;ine, definisan je sastav hranljive podloge za dobijanje maksimalnog sadržaja biomase P. stutzeri D1 (1 g/L glukoze, 3 g/l KNO₃ i 4 g/L peptone) i P. stutzeri ATCC 17588 (2 g/L KNO₃ i 4 g/L peptona). Osim toga, optimizovani su i odabrani bioprocesni parametri za proizvodnju biomase oba denitrifikatora &ndash; temperatura 30&deg;C, pH vrednost kultivacionog medijuma 7 jedinica i veličina inokuluma 5% (v/v). Validacija optimizovanih parametara kultivacije izvr&scaron;ena je izvođenjem nove serije ogleda pri definisanim uslovima u laboratorijskim bioreaktorima ukupnih zapremina 3 L i 7 L, čime je pokazana stabilnost bioprocesa prilikom uvećanja zapremine kultivacione tečnosti uz ostvarenje maksimalnog sadržaja biomase od 9 log CFU/ml za oba ispitivana soja uz minimalno opterećenje otpadnog toka. Rezultati primene denitrifikatora u vidu bioaktivnog sistema (pulpa sa suspenzijom denitrifikatora) koji je nano&scaron;en na povr&scaron;inu ve&scaron;tački kontaminiranih model supstrata opeke ukazuju na visoku efikasnost uklanjanja nitrata i to 95 % u slučaju P. stutzeri D1 i 72% u slučaju referentnog soja. Ispitivanjem bioaktivnih sistema primenjenih na kontaminirane uzorke stenskog materijala konstatovano je da se najveća efikasnost postiže tokom prvih 24 h, kada je efikasnost bioaktivnog sistema sa P. stutzeri ATCC 17588 47,7%, a bioaktivnog sistema sa P. stutzeri D1 čak 89,2%.<br />U okviru doktorske disertacije izolovanjem i karakterizacijom visoko efikasnog denitrifikatora P. stutzeri D1 i detaljnim prikazom postupka optimizacije bioprocesnih parametara i validacije optimizovanih bioprocesnih parametara u laboratorijskim bioreaktorima načinjeni su prvi koraci ka definisanju idejnog re&scaron;enja i uvećanju razmera predloženog bioprocesa. Takođe, visoka efikasnost odabranih denitrifikatora u postupcima bioči&scaron;ćenja različitih građevinskih materijala, koja je postignuta u laboratorijskim uslovima predstavlja dobar osnov za nastavak istraživanja u realnim uslovima, kako na građevinskim materijalima, tako i u tretmanima zemlji&scaron;ta i otpadnih voda.</p> / <p>The main goal of this PhD thesis is to establish the procedure of isolation and characterization of denitrifiers along with the optimization of biotechnological conditions for the biomass production and testing the efficiency of the produced biomass in terms of biocleaning of various building materials.<br />Within the research involved in this PhD thesis, novel denitrifier strain was isolated and identified as Pseudomonas stutzeri by using MALDI TOF. In all experiments, the characteristics and performances of the newly isolated strain of P. stutzeri (internally denoted as D1) were compared with the reference strain P. stutzeri ATCC 17588, which is a model microorganism for studying the denitrification process. By using PCR method the presence of four key denitrification genes (napA, nirS, norB and nosZ) in genome of the both strains was established indicating their genetic predisposition to perform complete aerobic denitrification. Also, the composition of the cultivation medium for obtaining the maximum content of biomass of P. stutzeri D1 (1 g/L glucose, 3 g/l KNO₃ and 4 g/L peptone) and P. stutzeri ATCC 17588 (2 g/L KNO₃ and 4 g/L peptone) was detemined by employing Box-Bhenken experimental design and RSM. In addition, the selected bioprocess parameters were optimized for the production of biomass of the both denitrifiers &ndash; temperature 30 &deg;C, pH value of the cultivation medium of 7 units and inoculum size of 5% (v/v). Validation of the optimized bioprocess parameters was carried out by performing new series of experiments under previously define conditions in laboratory bioreactors with total volumes of 3 L and 7 L. The results of validation showed the stability of bioprocess while increasing the volume of the cultivation broth with the achievment of a maximum biomass content og 9 log CFU/ml for the both strains with minimum load of waste stream. The results of the application of the denitrifiers as a part of bioactive system (poultice with a suspension of denitrifiers) used onto the artificially contaminated brick model supstrates indicate a high efficiency of nitrate removal, namely 95% in the case of P. stutzeri D1 and 72% in the case of reference strain. By examing bioactive systems applied to contaminated stone material it was found that the highest efficiency in nitrate removal was achieved during the first 24 h (47.7% for the reference strian and 89.2% for the P. stutzeri D1).<br />The results obtained in this PhD thesis including the established procedure for isolation and characterization of denitrifiers and detailed presentation of the optimization and validation process of bioprocess parameters represent the first steps made towards defining the conceptual solution and scale up of the proposed bioprocess. Moreover, the high efficiency of selected denitrificators in the biological treatment of various building materials, achieved in laboratory conditions is a good basis for continuing research in real conditions on construction materials, as well as in soil and wastewater treatment.</p>

In nature bacteria predominantly live on surfaces, in matrix-encased communities called biofilms. Biofilm formation displays dynamic developmental patterns resembling those of multicellular organisms. Using cooperative traits such as cell-cell signaling, bacteria in biofilms form complex architectures, known as microcolonies, in which cells become highly differentiated from their planktonic counterparts. Microcolonies are generally highly tolerant to bactericides, rendering biofilms extremely difficult to eradicate. The aim of this study was to investigate the last, and least understood stage of biofilm development, which involves the coordinated dispersal of single cells that revert to a free-swimming planktonic phenotype and escape from the biofilm. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. In the model organism Pseudomonas aeruginosa, reproducible patterns of cell death and dispersal can occur within biofilm structures, leaving behind empty or hollow microcolonies. These events were previously linked with the appearance of oxidative and/or nitrosative stress in mature microcolonies. Here, the involvement of reactive oxygen and nitrogen intermediates in biofilm development and dispersal processes was investigated in both mono- and mixed-species biofilms. By using specific fluorescent dyes and P. aeruginosa mutant strains, nitric oxide (NO), a by-product of anaerobic respiration and an important messenger molecule in biological systems, was found to play a major role in P. aeruginosa biofilm dispersal. Further, the results demonstrated that exposure to physiological, non-toxic concentrations of NO (in the low nanomolar range) causes biofilm dispersal in P. aeruginosa and restores its vulnerability to conventional antimicrobials. By using microarray techniques, NO was shown to induce global changes in genetic expression, including enhanced metabolic activity and motility and decreased adhesion and virulence in P. aeruginosa biofilms. The regulatory pathway implicated c-di-GMP, a newly discovered messenger molecule involved in the transition from sessility to motility in many bacterial species. NO-mediated dispersal was also observed in other single- and multi-species biofilms of clinically and industrially relevant organisms. Hence, the combined exposure to NO and bactericides was identified as a potential novel strategy for the removal of microbial communities, providing a low cost and environmentally safe solution to biofilm control.

Pseudomonas aeruginosa is an opportunistic human pathogen that infects immunocompromised individuals such as those suffering from burns or the genetic disorder cystic fibrosis. This organism utilizes a cell-cell communication mechanism known as quorum sensing (QS) to coordinate virulence gene expression and biofilm formation. It has three interconnected QS systems, namely las, rhl and pqs. Each system is comprised of autoinducer synthesis genes, lasI, rhlI, and pqsABCDH, and the cognate regulatory genes, lasR, rhlR, and pqsR, respectively. Here, we primarily focused on understanding the regulatory mechanisms of QS, which we investigated at two levels. First, we sought to identify additional activators that regulate QS at the level of the las and rhl systems, and second, we investigated the regulation of downstream genes, particularly biofilm exopolysaccharide genes, by QS. For the first approach, we employed a mutagenesis screen to identify global QS activators. We screened a non-redundant transposon library for mutants deficient in QS-dependent phenotypes. We identified a novel regulator, GidA, a glucose-inhibited cell division protein, that selectively controls QS gene expression posttranscriptionally via RhlR-dependent and –independent pathways. For the second part, we established a regulatory link between QS and Pel exopolysaccharide. We showed that the las system represses Pel and modulates colony biofilm structure through the pqs pathway. LasR mediated colony rugosity via 4-hydroxy-2-alkylquinolines in a PqsR-independent manner, ascribing a novel function to this class of signaling molecules in P. aeruginosa. Taken together, our study highlights the complexity of QS, which involves integration of various regulatory pathways to control downstream processes in response to different environmental conditions. / Graduation date: 2012

A major factor in the ability of Pseudomonas fluorescens Pf-5 to act as a biological control agent is its production of antibiotics, including pyoluteorin (PLT), 2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin (PRN). The data provided in this thesis demonstrate that the presence of any of these antibiotics in the extracellular milieu affects production of that same antibiotic, as well as others, by Pf-5. Amending the growth medium with antibiotics had multiple effects on secondary metabolism in Pf-5. i) PLT positively regulated its own production, ii) 2,4-DAPG positively regulated its own production. iii) PLT suppressed 2,4-DAPG production. iv) 2,4- DAPG inhibited PLT production. v) PLT suppressed transcription of a heterologous ferric-pyoverdine uptake gene. vi) PRN exerted a slight inhibitory effect on PLT gene transcription and production. PLT autoinduction by Pf-5 was extensively characterized, and was shown to require concentrations of exogenous PLT in the nanomolar range. These low concentrations are comparable to those of many molecules proposed to function in signaling roles. PLT served as a signal between distinct populations of cells within the rhizosphere, where it prompted autoinduction by those cells. Aside from effects of Pf- 5 antibiotics on one another, I also described the positive effect of exogenous PLT on expression of a set of transport genes flanking the PLT biosynthetic gene cluster. Sequence data and experimental evidence suggests that these genes encode a transport apparatus for PLT. The deduced amino acid sequences for four adjacent open reading frames together resemble Type I secretion apparatuses, which typically function in transport of proteins rather than secondary metabolites. The intact transporter genes are necessary for optimal PLT production. Taken together, the data from the studies described herein demonstrate that i) the production of PLT by Pf-5 can affect the production of PLT by neighboring cells, and ii) PLT and other exogenous secondary metabolites have both autoregulatory and cross-regulatory effects in culture. Because Pf-5 derivatives engaged in PLT crossfeeding in the rhizosphere, it is likely that cross-feeding occurs for other secondary metabolites as well. Thus, production of an antibiotic by one cell can profoundly affect secondary metabolism in neighboring cells occupying natural habitats. / Graduation date: 2004

Reproduction de : Thèse de doctorat : Physiologie, Biologie des organismes, populations, interactions : Lille 2 : 2008. / Résumé en français et en anglais. Titre provenant de l'écran-titre. Bibliogr. f. 143-175.

The Gram-negative bacterium Pseudomonas aeruginosa is a notorious opportunistic pathogen of individuals with the genetic disease cystic fibrosis (CF). Pseudomonas aeruginosa establishes a chronic infection within the CF lung, where the sputum accumulation characteristic of CF provides a complex and copious growth substrate. P. aeruginosa can grow to high densities in vivo (>10⁹ cells/ml lung sputum), and exacerbations associated with P. aeruginosa high density in vivo growth are primary contributors to CF morbidity and mortality. Surprisingly little is known about the catabolic processes that underlie P. aeruginosa in vivo growth. Unfortunately, nutritional modeling of the CF lung environment in animal models is difficult, as current animal models fail to mimic the sputum accumulation characteristic of CF. In this dissertation, I describe the use of expectorated CF sputum as a P. aeruginosa in vitro growth medium. Using global expression analysis, I show that P. aeruginosa up-regulates genes important for amino acid and lactate metabolism during growth in CF sputum as compared to a laboratory medium. P. aeruginosa also demonstrates enhanced production of the cell-cell communication signal 2-heptyl-3-hydroxy-4-quinolone (the Pseudomonas quinolone signal, PQS), a critical regulator of virulence factor production, during growth in CF sputum. Further, I use chemical analyses of CF sputum samples to develop a defined, synthetic medium that can be used to nutritionally model in vivo conditions. Using this medium, I show that PQS biosynthesis and aromatic amino acid metabolism are intimately linked and that cell-cell communication mediated by PQS is strikingly dependent upon the growth environment of P. aeruginosa. In addition, I demonstrate that P. aeruginosa preferentially consumes specific carbon sources present in the CF sputum milieu during rapid growth. I also describe the use of in vivo-relevant nutrient concentrations to evaluate the potential for P. aeruginosa anaerobic growth in CF sputum. Finally, I describe the purification and characterization of the aromatic amino acid-responsive transcriptional regulator PhhR and discuss its potential role in regulation of P. aeruginosa in vivo carbon substrate preference. / text

This work was undertaken with the objective of isolating and characterising the bdh gene of P. aeruginosa PAOI. Isolation of the bdh gene was initially attempted by PCR amplification and then by heterologous complementation of E. coli (LS5218) and S. meliloti (Rm11107) strains unable to catabolise 3-hydroxybutyrate. Three classes of plasmids were isolated. Class I comprised two plasmids, p5218-02 and p5218-07, isolated via complementation of LS5218, which were capable of complementing both LS5218 and Rm11107 for growth on 3-hydroxybutyrate. 3-hydroxybutyrate dehydrogenase (BDH) activity was not detected in an extract of LS5218 (p5218-02). The sole 3.6-kb EcoRI fist was partially sequenced and found to have three putative open reading frames (ORF). ORF 1 is homologous to the fusE gene of E. coli. We hypothesised that p5218-02 encodes an enzyme capable of degrading 3 hydroxybutyrate, but does not encode the bdh gene. Plasmids of class II (p30065) and class III (p30066) were isolated via complementation of Rm11107. Significant BDH activity was detected in an extract of Rm11107 (p30066), but not in Rm11107, leading to the hypothesis that p30066 carries the bdh gene.

Disertacijos tema: Potencialių hospitalinės pneumonijos sukėlėjų Pseudomonas aeruginosa ir Klebsiella pneumoniae patogeniškumo veiksniai bei jų įtaka ligos eigai Darbo tikslas -ištirti Pseudomonas aeruginosa ir Klebsiella pneumoniae padermių, kolonizavusių apatinius kvėpavimo takus ar sukėlusių hospitalinę pneumoniją, patogeniškumo veiksnius ir jų įtaką hospitalinės pneumonijos eigai. Uždaviniai: • Ištirti hospitalinę pneumoniją sukėlusių ar apatinius kvėpavimo takus kolonizavusių Pseudomonas aeruginosa padermių patogeniškumo veiksnius - atsparumą serumo baktericidiniam poveikiui, gebėjimą įsiskverbti į kvėpavimo takų epitelio ląsteles, atsparumą antibiotikams ir O serogrupinę priklausomybę. • Įvertinti Pseudomonas aeruginosa padermių patogeniškumo veiksnių tarpusavio sąsajas. • Ištirti Klebsiella pneumoniae padermių, sukėlusių hospitalinę pneumoniją ar kolonizavusių apatinius kvėpavimo takus, gebėjimą gaminti plataus spektro beta laktamazes bei atsparumą antibiotikams. • Įvertinti Pseudomonas aeruginosa ir Klebsiella pneumoniae padermių patogeniškumo veiksnių įtaką hospitalinės pneumonijos eigai. Darbas yra pirmas Lietuvoje, kurio metu ne tik nustatytas Pseudomonas aeruginosa patogeniškumo veiksnys – atsparumas serumo baktericidiniam poveikiui, bet ir įvertinta galima šį patogeniškumo veiksnį įgijusių Pseudomonas aeruginosa padermių įtaka hospitalinės pneumonijos vystytis bei ligos eigai. Pirmą kartą apskritai vertintas Pseudomonas aeruginosa padermių gebėjimas įsiskverbti į... [toliau žr. visą tekstą] / The aim of the study: To examine pathogenicity factors of Pseudomonas aeruginosa and Klebsiella pneumoniae strains, colonizing lower respiratory tract or causing hospital-acquired pneumonia, and to evaluate their influence on the course of hospital-acquired pneumonia. Objectives of the sudy: 1. To examine pathogenicity factors – resistance to serum bactericidal activity, ability to penetrate epithelial cells of the respiratory tract, dependence of O serogroup, and resistance to antibiotics – of Pseudomonas aeruginosa strains, colonizing lower respiratory tract or causing hospital-acquired pneumonia. 2. To evaluate the relationship between pathogenicity factors of Pseudomonas aeruginosa strains. 3. To examine the ability of Klebsiella pneumoniae strains, colonizing lower respiratory tract or causing hospital-acquired pneumonia, to produce extended-spectrum beta-lactamases and resistance of these pathogen to antibiotics. 4. To evaluate the influence of pathogenicity factors of Pseudomonas aeruginosa and Klebsiella pneumoniae strains on the course of hospital-acquired pneumonia. Such work is first in Lithuania, because we determined not only pathogenicity factors of Pseudomonas aeruginosa – i.e., resistance to bactericidal activity of serum, but also evaluated possible influence of Pseudomonas aeruginosa strains, having this pathogenicity factor, on hospital-acquired pneumonia development and outcome. Therefore, the ability of Pseudomonas aeruginosa strains to invasive into... [to full text]

In nature bacteria predominantly live on surfaces, in matrix-encased communities called biofilms. Biofilm formation displays dynamic developmental patterns resembling those of multicellular organisms. Using cooperative traits such as cell-cell signaling, bacteria in biofilms form complex architectures, known as microcolonies, in which cells become highly differentiated from their planktonic counterparts. Microcolonies are generally highly tolerant to bactericides, rendering biofilms extremely difficult to eradicate. The aim of this study was to investigate the last, and least understood stage of biofilm development, which involves the coordinated dispersal of single cells that revert to a free-swimming planktonic phenotype and escape from the biofilm. Strategies to induce biofilm dispersal are of interest due to their potential to prevent biofilms and biofilm-related infections. In the model organism Pseudomonas aeruginosa, reproducible patterns of cell death and dispersal can occur within biofilm structures, leaving behind empty or hollow microcolonies. These events were previously linked with the appearance of oxidative and/or nitrosative stress in mature microcolonies. Here, the involvement of reactive oxygen and nitrogen intermediates in biofilm development and dispersal processes was investigated in both mono- and mixed-species biofilms. By using specific fluorescent dyes and P. aeruginosa mutant strains, nitric oxide (NO), a by-product of anaerobic respiration and an important messenger molecule in biological systems, was found to play a major role in P. aeruginosa biofilm dispersal. Further, the results demonstrated that exposure to physiological, non-toxic concentrations of NO (in the low nanomolar range) causes biofilm dispersal in P. aeruginosa and restores its vulnerability to conventional antimicrobials. By using microarray techniques, NO was shown to induce global changes in genetic expression, including enhanced metabolic activity and motility and decreased adhesion and virulence in P. aeruginosa biofilms. The regulatory pathway implicated c-di-GMP, a newly discovered messenger molecule involved in the transition from sessility to motility in many bacterial species. NO-mediated dispersal was also observed in other single- and multi-species biofilms of clinically and industrially relevant organisms. Hence, the combined exposure to NO and bactericides was identified as a potential novel strategy for the removal of microbial communities, providing a low cost and environmentally safe solution to biofilm control.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2008. / Bibliography: p. 257-274. / 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. / 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. / Mode of access: World Wide Web. / xiii, 274 p. ill