Please read the abstract (Summary) in the section, 00front, of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Pseudomonas aeruginosa, a ubiquitous environmental bacterium and an opportunistic human pathogen, forms biofilms through a series of interactions between the cells and adherence to surfaces. Not only does rhamnolipid contribute to the pathogenic potential of P. aeruginosa, but it has also been reported that the bacterium utilises rhamnolipid to actively maintain the void spaces surrounding microcolonies, thus contributing to the architecture of P. aeruginosa biofilms. The P. aeruginosa rhlAB operon encodes the enzyme rhamnosyltransferase I, which produces mono-rhamnolipid, and the induction of rhlAB is dependent on the quorum sensing transcription activator RhIR complexed with the auto inducer N-butyryl-homoserine lactone. In this study, several aspects related to rhamnolipid biosynthesis and regulation in P. aeruginosa PAO1 were investigated. As a first step, a biochemical assay was developed and optimised whereby the concentration of rhamnolipid could be accurately quantified following its extraction from small sample volumes. Although the optimised rhamnolipid assay is not able to distinguish between different rhamnolipids or between different homologs of a specific rhamnolipid, it is, however, simple to perform, cost¬effective and does not rely on the use of specialised equipment. Subsequently an rhlAB-deficient mutant strain of P. aeruginosa PAOI strain was generated. For this purpose, three allelic exchange strategies, i.e. plasmid incompatibility, the use of a SacB counter-selectable marker and a combination of these approaches, were investigated by making use of newly constructed allelic exchange vector systems. The results that were obtained indicated that, of the three approaches, the latter was most efficient in generating the desired P. aeruginosa mutant strain, and 90% of the derived strains were found to be double reciprocal mutants. Reporter gene technology, using the genes encoding for stable and unstable variants of the green fluorescent protein (GFP), was finally used to investigate the transcriptional activity of the rhlA promoter in P. aeruginosa biofilms under conditions of continuous flow using glass as substratum. For this purpose, mini-CTX-GFP reporter vectors, containing stable and unstable variants of the gfp reporter gene, were constructed that allow for integration of a single copy of the transcriptional fusion in a defined, non-essential region onto the P. aeruginosa genome. Several global regulators have been reported to playa role in regulating quorum sensing and/or rhamnolipid biosynthesis in P. aeruginosa, amongst other, the sigma factors RpoS and RpoN. Therefore, rhlA promoter activity was also investigated in biofilms of P. aeruginosa strains lacking either RpoN or RpoS. Although structural differences between the biofilms formed by the P. aeruginosa wild-type PAD 1 and respective mutant strains were noted, transcription of rhlA appeared to be constitutive from 24 h onwards and did not appear to be localised to specific areas within the microcolonies or biofilms. These results, combined with those obtained by batch analysis, indicated that RpoS positively regulates rhlA transcription, whilst RpoN did not appear to influence rhlA promoter activity under the conditions used in this study. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is one of a group of bacteria known as cyanotrophs that exhibit the unique ability to grow on toxic cyanide as the sole nitrogen source. This ability has previously been genetically linked to a conserved cluster of seven genes (Nit1C), the signature gene (nitC) coding for a nitrilase enzyme. Nitrilases convert nitriles to ammonia and a carboxylic acid. Still, for the Pf11764 NitC enzyme (Nit11764), no in vivo substrate has been identified, and the basis of the enzyme's requirement for cyanide growth has remained unclear. Therefore, the gene was cloned and the enzyme was characterized with respect to its structure and function. These efforts resulted in the unique discovery that, aside from its nitrilase activity, Nit11764 exhibits nuclease activity towards both DNA and RNA. This ability is consistent with computer analysis of the protein providing evidence of a preponderance of amino acids with a high probability for RNA binding. A Nit11764 knock-out mutant was shown to exhibit a higher sensitivity to both cyanide (KCN) and mitomycin C, both known to induce chromosomal damage. Thus, the overall conclusion is that Nit11764, and likely the entire Nit1C gene cluster, functions as a possible repair mechanism for overcoming the damage inflicted on Pf11764 nucleic acids by toxic cyanide. Towards a further investigation of the Nit1C gene cluster in Pf11764, a second gene (nitH) annotated as a monooxygenase was also investigated. Interestingly, computer-based analysis shows that NitH also harbors a preponderance of RNA-binding amino acids. The nitH gene was cloned into an expression vector with the long-range goal of defining its role in CN utilization.

A study was conducted to find the effect of magnesium, calcium, manganese and zinc ions on the infection of Psudomonas aeruginosa strain 1C by RNA bacteriophage 7s. When an 18 hour progeny experiment was performed, it was found that magnesium, calcium and manganese had different effects on bacteriophage production and was dependent on the bacterial growth conditions. RNA bacteriophage 7s progeny production was significantly enhanced by the addition of magnesium to cultures of Psudomonas aeruginosa 1C grown in a magnesium deficient medium. Under these environmental conditions there was a slight increase in progeny in the presence of calcium. When Psudomonas aeruginosa 1c was grown in a complete medium, the infection of cells by bacteriophage 7s was enhanced by magnesium and calcium but not manganese or zinc, as demonstrated by the One Step Growth Curve.

El uso de biosurfactantes en biorremediación facilita y acelera la degradación microbiana de hidrocarburos. El método del agar CTAB/MB creado por Siegmund y Wagner para el screening de cepas productoras de ramnolípidos (RL), ha sido ampliamente utilizado sin sufrir mejoras significativas en más de 20 años. Con el fin de optimizar la técnica como método cuantitativo, se hicieron placas con agar CTAB/MB y se probaron diferentes variables, tales como tiempo de incubación, refrigeración, concentración de CTAB, presencia de azul de metileno, diámetro de los pozos y volumen del inóculo. Adicionalmente, se desarrolló un nuevo método para detectar el RL de los halos mediante precipitación con HCl, lo cual permite la observación de un nuevo patrón de halos más fáciles de observar y medir. Este trabajo reafirma que este método no es del todo apropiado para un análisis cuantitativo fino, debido a la dificultad de correlacionar de forma precisa la concentración de RL y el área de los halos formados. La difusión del RL no parece tener un comportamiento simple y existen numerosos factores que afectan la velocidad de migración del RL. A pesar de todo, aún es muy útil para una pre-selección semi-cuantitativa de cepas productoras de RL; si bien no es posible determinar exactamente cuánto RL se ha producido, aún es posible diferenciar cepas con productividad significativamente diferentes bajo condiciones similares. La aplicación de este método y su modificación permitió reducir el número candidatos de 2517 cepas potenciales a tan solo 3 cepas superproductoras (0.12%). Palabras clave: Bromuro de Cetiltrimetilamonio (CTAB), azul de metileno, Pseudomonas, screening, ramnolípidos, biosurfactantes / Tesis

Opportunistic pathogens (e.g., Legionella pneumophila, Mycobacterium avium complex, Acanthamoeba polyphaga, Pseudomonas aeruginosa) residing in human-made water systems, particularly premise plumbing, are now the primary source of water-borne disease in developed countries. The prevention and control of opportunistic pathogens is a new challenge in premise plumbing due to the limited knowledge concerning the factors driving their occurrence and regrowth mechanisms, and also the complexity of premise plumbing conditions. The goal of this study is to identify key factors governing occurrence of opportunistic pathogens in drinking water distribution systems, particularly premise plumbing, via field investigations and lab-scale experiments. A molecular survey of three opportunistic pathogens (L. pneumophila, M. avium, P. aeruginosa), related groups (Legionella and mycobacteria) and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis) was performed in two real-word chloraminated drinking water distribution systems using quantitative polymerase chain reaction (q-PCR). A high occurrence of Legionella (" 69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (" 20%) and M. avium (" 33.3%), and rare detection of Pseudomonas aeruginosa (" 13.3%) was observed in both systems. Hartmanella vermiformis was more prevalent than Acanthamoeba. Three-minute flushing resulted in reduced gene copies of Legionella, mycobacteria, H. vermiformis and 16S rRNA genes (P<0.05) and distinct microbial community structure in postflushing water, implying strong regrowth potential of opportunistic pathogens in premise pluming. In order to examine the influence of pipe material, disinfectant type, and water age on occurrence and persistence of the target microorganisms, triplicate simulated distribution systems (SDSs) comparing iron, cement and PVC pipe materials were fed either chlorinated or chloraminated tap water, and were sampled at water ages ranging from 1d to 5.7d. q-PCR quantification of target microorganisms in both biofilm and bulk water revealed that Legionella, mycobacteria, P. aeruginosa and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. Influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Natural colonization of Legionella spp., Mycobacterium spp., Acanthamoeba spp., H. vermiformis and M. avium was also observed in biofilms from five annular reactors, which were used to investigate effects of prior granular activated carbon (GAC) biofiltration and disinfectant type (chlorine, chloramine) on opportunistic pathogens under premise plumbing conditions. GAC pre-treatment effectively reduced total organic carbon (TOC). In most cases, total bacteria and opportunistic pathogens were higher in undisinfected annular reactors, but the levels were not proportional to the level of GAC pre-treatment/TOC. Chlorine was more effective for controlling mycobacteria and Acanthamoeba, whereas chloramine was more effective for controlling Legionella. Both chlorine and chloramine effectively reduced M. avium and H. vermiformis numbers. Pyrosequencing of 16S rRNA genes in biofilms revealed a significant effect of GAC pre-treatment and disinfectant type on the microbial community structure. Overall, the study provides insights to critical factors triggering proliferation of opportunistic pathogens in drinking water systems. Knowledge gained from this study can assist in formulating practical guidance for drinking water utilities and water consumers in terms of opportunistic pathogen prevention and control. / Ph. D.

No description available.

Type IV pili (T4P) are hair-like adhesins involved in many processes, including surface attachment, twitching, DNA uptake, electron transfer, and pathogenesis. These flexible filaments are expressed in various pathogens, including the opportunistic pathogen Pseudomonas aeruginosa. The pilus fibre is primarily composed of the major pilin structural subunit, PilA, which is rapidly polymerized or depolymerized during pilus extension or retraction, respectively. The transcription of pilA is tightly controlled by the PilS-PilR two-component system, which responds to fluctuating levels of PilA in the inner membrane. In addition to pilA, the response regulator, PilR, also regulates a subset of other non-T4P related genes. Here, we used hyperactivating point mutants in the PilS-PilR two-component system, which induce hyperpiliation without loss of pilus function, to assess the effects of increased surface pili expression on virulence against Caenorhabditis elegans, and to identify additional non-T4P genes regulated by the PilS-PilR two-component system. We hypothesized that dysregulation of the PilS-PilR two-component system impacts the expression of pilA and other genes, which impacts both surface piliation and T4P dynamics, resulting in altered P. aeruginosa virulence. C. elegans slow killing assays revealed that hyperpiliation, independently of T4P function, reduces virulence of model P. aeruginosa strains PAK and PA14. We propose a model whereby a surfeit of pili reduces virulence, potentially through impeding effective engagement of contact-dependent antagonism systems, such as the type III secretion system. Transcriptomic analysis of the hyperactive PilR point mutant also identified a subset of 26 genes, including those related to phenazine biosynthesis, quorum sensing, and ethanol oxidation, regulated by the PilS-PilR two-component system. Last, a T4P cysteine-labelling system was implemented for P. aeruginosa, allowing for the visualization of real-time pilus dynamics. Together, this work provides new insights into the consequences of hyperpiliation and the scope of the PilS-PilR signalling network, as well as novel tools for investigating P. aeruginosa T4P dynamics in vivo. / Thesis / Master of Science (MSc) / Pseudomonas aeruginosa is a major contributor to hospital-acquired infections and is of particular concern due to its intrinsic resistance to many frontline antibiotics. To aid in infection, Pseudomonas encodes an arsenal of virulence factors, including type IV pili (T4P), hair-like adhesins involved in many processes, such as twitching motility and surface attachment. T4P are primarily composed of the major pilin, PilA, whose expression is tightly regulated by the PilS-PilR two-component system. The sensor kinase, PilS, monitors the inner membrane PilA inventory and modifies activity of the response regulator, PilR, to regulate pilA transcription. Here, we demonstrate that P. aeruginosa virulence in a roundworm infection model is reduced when the amount of T4P expressed at the cell surface increases, regardless of the ability of the bacteria to twitch. We propose that inappropriate increases in surface T4P expression may impair pathogenicity-associated systems which require intimate host-cell contact. New genes in the regulon of the PilS-PilR two-component system were also identified. A tool to fluorescently label and image T4P in real-time using microscopy was established in the lab. This work highlights the consequences of increased surface T4P expression, providing potential new targets for antipseudomonal therapeutics which act on components involved in T4P expression and function.

No description available.

Type IV pili (T4P) are long, fibrous surface appendages involved in attachment, motility, biofilm formation and DNA uptake that are expressed by bacteria and archaea. They are an important virulence factor for a number of bacteria, including Pseudomonas aeruginosa, an opportunistic pathogen that is a common cause of nosocomial infections. T4P are composed mainly of monomers of the major pilin subunit, PilA, although several low abundance proteins called minor pilins are also present. These surface-exposed proteins are potential vaccine candidates, although a more complete understanding of their diversity and function is required for the rational development of a pilus-based vaccine. There are five distinct groups of P. aeruginosa major pilins, which vary based on their sequence and their associated accessory proteins, and two distinct sets of minor pilins, although the roles of the latter in pilus biology are poorly understood. This study focuses on the structural characterization of major and minor pilins and functional implications for pilus assembly and disassembly dynamics. The structural analysis of major pilins from groups III and V revealed specific differences in pilin structure that may affect subunit interactions within the pilus fibre and interactions with their specific accessory proteins and minor pilins. The minor pilins PilVWX were shown to form a putative subcomplex with the adhesin and anti-retraction protein PilY1, which is proposed to prime pilus assembly and thus traffic PilY1 to the bacterial surface. High resolution X-ray crystal structures of the minor pilins FimU and PilE were solved and functional characterization suggested that FimU and PilE are necessary for efficient pilus assembly to stably connect the priming subcomplex to the major pilin subunits. Together, this work has increased our understanding of pilin diversity and defined a concrete role for the minor pilins in pilus assembly. / Thesis / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is a bacterium that can take advantage of a weakened immune system to cause lethal infections. The first step of infection involves attachment to the host using long sticky fibres called type IV pili. Each fibre is composed primarily of a single protein, the major pilin, but also contains low abundance proteins called minor pilins. Without these proteins, the bacteria can’t attach and cause infections, making pilins excellent vaccine candidates. This study focused on the characterization of major and minor pilins to understand the diversity of these proteins and how these differences might affect pilus assembly. We show that the molecular structure of the major pilin differs between strains although the core architecture is the same, and that the minor pilins are required for initiation of pilus assembly. This work furthers our understanding of the structures and functions of pilin proteins, and provides information helpful for the development of vaccines.